ISSN 0012-4966, Doklady Biological Sciences, 2016, Vol. 470, pp. 224–227. © Pleiades Publishing, Ltd., 2016. Original Russian Text © S.V. Kalemenev, O.E. Zubareva, V.V. Sizov, V.V. Lavrent’eva, N.Ya. Lukomskaya, K.Kh. Kim, A.V. Zaitsev, L.G. Magazanik, 2016, published in Doklady Akademii Nauk, 2016, Vol. 470, No. 6, pp. 732–735.
PHYSIOLOGY
Memantine Attenuates Cognitive Impairments after Status Epilepticus Induced in a Lithium–Pilocarpine Model S. V. Kalemeneva, O. E. Zubarevaa,b, V. V. Sizovb, V. V. Lavrent’evaa, N. Ya. Lukomskayaa, K. Kh. Kima, A. V. Zaitseva, and Corresponding-Member of the RAS L. G. Magazanika,c,* Received May 31, 2016
Abstract—The capability of memantine, a noncompetitive antagonist of the NMDA receptors, to prevent impairments of cognitive functions in rats was investigated in the lithium–pilocarpine model of epilepsy. After status epilepticus, rats exhibited impaired exploratory behavior and spatial memory, and a decline of extinction of orienting behavior. Memantine administration prevented these disturbances. Thus, the blockade of the NMDA receptors immediately after status epilepticus allowed prevention of the development of the possible cognitive impairments. DOI: 10.1134/S0012496616050148
Epilepsy is one of widely distributed neurological disorders of humans, which often results in cognitive impairments [1]. Difficulties in the treatment of this pathology are related to its heterogeneous nature and a variety of pathogenetic mechanisms, which are mostly associated with an imbalance between the inhibitory (GABA) and excitatory (glutamate) influences on neurons of different brain regions [2]. The excessive release of glutamate probably induces molecular modifications and neurodegeneration, which are followed by impairments of cognitive functions [3–6]. These processes are largely mediated by the NMDA type of glutamate receptors [7]. The capability to use antagonists of the NMDA receptors for correction of post-seizure psychoneurological impairments is being widely discussed [1, 2], although the therapeutic potential of specific drugs is poorly studied. In the present study, the capability of memantine, a noncompetitive antagonist of the NMDA receptors, to prevent mortality and impairments of cognitive functions in rats was investigated in the lithium–pilocarpine model of epilepsy. This model allows the reproduction of pathological processes observed in human temporal epilepsy [8], including epilepsyinduced memory impairments [4, 9]. The experiments were performed on 1.5-monthold male Wistar rats in accordance with the Protocol a Sechenov
Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia b Institute of Experimental Medicine, St. Petersburg, Russia c St. Petersburg State University, St. Petersburg, Russia * e-mail:
[email protected]
for Experiments with Laboratory Animals of the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences. Status epilepticus was induced using the lithium–pilocarpine model [8]. One day prior to intraperitoneal (i.p.) administration of pilocarpine (Sigma-Aldrich, United States) at a dose of 30 mg/kg, the rats were i.p. injected with LiCl (Sigma-Aldrich, United States) at a dose of 127 mg/kg. In order to prevent excessive activation of peripheral muscarinic receptors, the rats were i.p. injected with methylscopolamine (Sigma-Aldrich, United States) at a dose of 1 mg/kg 1 h prior to the LiCl administration. The control animals were injected with LiCl dissolved in sterile isotonic saline solution at the same time points. All animals exhibited convulsions after the pilocarpine treatment. Three hours after the pilocarpine administration, some animals (the treated group) were i.p. injected with memantine (Sigma-Aldrich, United States) at a dose of 10 mg/kg whereas the other rats (the untreated group) were injected with isotonic saline. Animal behavior was evaluated 8–15 days after the pilocarpine administration. Orienting and exploratory behavior in a new environment was studied for 5 min in the open field test (OFT). In order to estimate the extinction of orienting and exploratory behavior, the repeated tests were performed for three consecutive days at 24-h intervals. All experiments were recorded using a video camera, and then, the ethological indices including exploration, anxiety, and locomotion were analyzed using the Fild-1 and Tracking software developed at the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences.
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Indices of rat behavior in the open field test Control (n = 17)
Untreated (n = 24)
Treated (n = 28)
Indices Rearing with climbing, duration, s Duration of exploration of a hole, s Number of groomings Grooming duration, s Rearing without climbing Duration of locomotion, s Distance traveled, m
Day 1
Day 2
18.7 ± 2.0
11.3 ± 1.7
Day 3
Day 1
12.5 ± 1.9 12.8 ± 1.2*
Day 2
Day 3
Day 1
Day 2
Day 3
9.7 ± 1.5
10.8 ± 1.1
16.3 ± 1.5
11.8 ± 1.7
8.3 ± 1.2
0.80 ± 0.04 0.82 ± 0.04 0.81 ± 0.07 0.64 ± 0.03* 0.65 ± 0.06 0.84 ± 0.05 0.89 ± 0.09# 0.74 ± 0.07 0.73 ± 0.08
7.8 ± 1.3
11.2 ± 1.8
9.1 ± 1.2
6.9 ± 1.3
7.8 ± 1.3
16.9 ± 2.3
35.6 ± 3.8
42.0 ± 4.6
16.5 ± 2.9
24.4 ± 4.2
6.1 ± 1.3
2.6 ± 1.0
4.3 ± 1.3
7.0 ± 1.7
6.5 ± 1.5
31.9 ± 1.9
19.8 ± 1.8
7.2 ± 0.4
5.2 ± 0.4
10.1 ± 1.1 12.1 ± 1.6#
12.0 ± 1.0 13.9 ± 1.3*
36.1 ± 3.9 37.1 ± 6.7*# 46.2 ± 5.5# 45.7 ± 4.6 7.1 ± 1.3
7.3 ± 1.2
4.6 ± 1.1
3.6 ± 0.8
19.9 ± 2.2 25.5 ± 1.3* 18.7 ± 2.0
22.7 ± 1.7
26.2 ± 2.0
18.9 ± 2.1
14.1 ± 1.5#
4.9 ± 0.4
5.3 ± 0.4
6.9 ± 0.4
5.0 ± 0.5
4.5 ± 0.4
6.9 ± 0.4
5.3 ± 0.5
Significant differences between the groups within the same experimental day are indicated with boldface type. Here and in the figure: data are presented as M ± m; * p < 0.05 and # p < 0.05, significant differences from the control or untreated rats, respectively; n, number of animals per group.
Spatial memory was studied in the Morris water maze (MWM) [10]. Training in MWM was started one day after the end of the OFT and repeated for four days. The training consisted of four trials. Shortening of the path length navigated by the animal in order to find the platform hidden under water, was used as an index of training efficiency. The distribution of the indices studied corresponded to normal according to the Kolmogorov– Smirnov test; therefore, we used parametrical statistical methods for analysis. Equality of the variances in the groups was estimated using Levene’s test. Oneway analysis of variances or Welch’s test for samples with unequal variances was used for evaluation of between group effects in specific experimental trials. Two-way analysis of variances was used for evaluation of the effects of drugs on the extinction of orienting and exploratory behavior in the OFT and task acquisition in the MWM. Post hoc comparisons for groups with equal and unequal variances were performed using Tukey’s test or the Games–Howel test, respectively. The differences were considered as significant at p ≤ 0.05. We found that memantine administration did not influence animal survival after status epilepticus. Within one week after the seizure induction, 31.7% of animals died in the memantine-treated group and DOKLADY BIOLOGICAL SCIENCES
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38.5% of rats died in the untreated group (p > 0.05 according to the ϕ* test). The untreated rats exhibited impaired exploratory behavior in the OFT (table). On the first testing day, the durations of rearing with climbing and exploration of holes were 32% and 20%, respectively, shorter as compared to the control animals (F2.66 = 3.26, p < 0.05 and F2.65 = 6.70, p < 0.01, respectively). Memantine treatment prevented these impairments. The duration of locomotion was shorter in the untreated as compared to control rats (F2.63 = 4.01, p < 0.05) and memantine administration did not influence this index. The untreated animals did not exhibit any impairment in the exploratory behavior on the second and third days of testing in the OFT. Thus, cognitive impairments induced by status epilepticus were more evident in the new environment. To analyze the development of extinction after repeated placement of a rat into the arena of the OFT, two-way analysis of variances was applied. We found the differences between the groups in the number and duration of rearing with climbing (F4.122 = 4.49, p < 0.01 and F4.122 = 2.76; p < 0.05, respectively) and in the duration of locomotion (F4.124 = 5.02; p < 0.01). The extinction of the orienting behavior was slower in the
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cm 2500 2000 1500
cm 800 600 400 200 0 Trials
Day 2
Day 3
cm 400
*#
300 200 100
1
2
3
4
0 Trials
1
2
3
4
1000 500 0 Trials 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Day 1 Day 2 Day 3 Day 4
Control PC PC+MEM
Distance navigated by rats in the MWM for finding of the hidden platform in the consecutive training days. Number of animals in the groups: control n = 17; pilocarpine (PC) n = 24; pilocarpine + memantine (PC+MEM) n = 28.
untreated rats as compared to both control and memantine-treated animals. Memantine treatment increased anxiety in the rats observed in the OFT. On the first testing day, the duration and number of groomings increased in this group of rats as compared to the control and untreated animals (F2.63 = 4.24, p < 0.05 and F2.64 = 3.52, p < 0.05, respectively). Similar changes were observed on the other days of testing (table). However, the other index of anxiety, rearing without climbing, was not changed. We did not find any differences in the anxiety indices studied in the group of untreated animals that survived seizures. Task acquisition in the MWM was observed in all groups of animals (figure); however, some differences in the indices of training were found. First trials to find the platform hidden under water of the second and other days of training are of specific interest because they allow us to consider the properties of long-term memory in animals. The elongation of the distance navigated, which indicated impaired long-term memory, was observed in the untreated animals on the second and third days of training (F2.58 = 3.80, p < 0.05 and F2.58 = 4.44, p < 0.05, respectively). Memantine treatment completely prevented these impairments. The lithium–pilocarpine model used in the present study allows the reproduction of not only seizures, but also cognitive impairments associated with temporal epilepsy in animals. Specifically, inmpaiments of spatial memory, behavior in a new environment, and extinction of orienting-exploratory response are observed in experimental animals [9, 11]. These data are in accordance with our results from the testing of the untreated rats. A possibility of use of the antagonists of the NMDA receptors for correction of cognitive deficit induced by seizures is now widely discussed [1]. Memantine, which is used for treatment of Alzheimer’s disease and some other neuropsychiatric distur-
bances, is considered among other drugs [12]. Our data show that the use of memantine immediately after the induction of status epilepticus allows complete prevention of cognitive impairments in rats. However, we revealed the anxiogenic side effect of memantine. Data on the effect of memantine on psychoemotional state of animals are controversial. Some authors have reported on the anxiogenic memantine effect [13], whereas others have demonstrated an anxiolytic effect of memantine [14]. These controversies in the results may be associated with the use of different doses of the drug or different behavioral tests. The effects of memantine on the psychoemotional state after seizures need additional studies. This is specifically important because increased anxiety and development of depression are often observed in patients with temporal epilepsy [15]. Thus, our study has demonstrated that the use of memantine for correction of cognitive impairments under the conditions of temporal epilepsy is very promising. However, search for new drugs without any side effects has to be continued. ACKNOWEDGMENTS This study was supported by the Russian Science Foundation, project no. 16-15-10202. REFERENCES 1. Leeman-Markowski, B.A. and Schachter, S.C., Neurol. Clin., 2016, vol. 34, pp. 183–204. 2. Lason, W., Chlebicka, M., and Rejdak, K., Pharmacol. Rept., 2013, vol. 65, pp. 787–801. 3. Zaitsev, A.V., Kim, K.K., Vasilev, D.S., et al., J. Neurosci. Res., 2015, vol. 93, pp. 454–465. 4. Kalemenev, S.V., Zubareva, O.E., Frolova, E.V., et al., Dokl. Biol. Sci., 2015, vol. 463, no. 3, pp. 175–177. 5. Malkin, S.L., Amakhin, D.V., Veniaminova, E.A., et al., Neuroscience, 2016, vol. 327, pp. 146–155. DOKLADY BIOLOGICAL SCIENCES
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MEMANTINE ATTENUATES COGNITIVE IMPAIRMENTS 6. Curia, G., Lucchi, C., Vinet, J., et al., Curr. Med. Chem., 2014, vol. 21, pp. 663–688. 7. Rice, A.C., Floyd, C.L., Lyeth, B.G., et al., Epilepsia, 1998, vol. 39, pp. 1148–1157. 8. Curia, G., Longo, D., Biagini, G., et al., J. Neurosci. Meth., 2008, vol. 172, pp. 143–157. 9. Lopes, M.W., Lopes, S.C., Santos, D.B., et al., Epilepsy Behav., 2016, vol. 55, pp. 92–100. 10. Trofimov, A.N., Zubareva, O.E., Simbirtsev, A.S., and Klimenko, V.M., Ros. Fiziol. Zh. im. I.M. Sechenova, 2012, vol. 98, pp. 782–792.
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11. Wolf, D.C., Bueno-Junior, L.S., Lopes-Aguiar, C., et al., Neuroscience, 2016, vol. 312, pp. 86–98. 12. Lipton, S.A., NeuroRx, 2004, vol. 1, pp. 101–110. 13. Zimmer, E.R., Torrez, V.R., Kalinine, E., et al., Front. Cell Neurosci., 2015, vol. 9, p. 219. 14. Kos, T. and Popik, P., Behav. Pharmacol., 2005, vol. 16, pp. 155–161. 15. Swinkels, W.A., Kuyk, J., van Dyck, R., and Spinhoven, P., Epilepsy Behav., 2005, vol. 7, pp. 37–50.
Translated by M. Stepanichev
