Brahz Research, 590 (1992) 48-52 ~:~ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
48
BRES 18044
Protective effects of serotonin reuptake inhibitors, citalopram and clomipramine, against hippocampal CA1 neuronal damage following transient ischemia in the gerbil Naoki N a k a t a , H i r o y u k i K a t o a n d Kyuya K o g u r e Department of Neurology, hastitute of Brain Diseases, Tohoku Unit'ersity School of Medicine, Sendai (Japan) (Accepted 7 April 1992)
Ke.v words: Cerebral ischemia; Serotonin; Citalopram; Clomipramine; Glutamate; Excitatory amino acid; Microdialysis; Gerbil
To clarify the role of serotonin in cerebral ischemia, we examined the effects of selective serotonin reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage in the gerbil. Pretreatment with citalopram (4() mg/kg i.p.) and clomipramine (20 mg/kg i.p.) protected against neuronal destruction of hippocampal CAI pyramidal cells following 5 rain of forebrain ischemia. Furthermore, microdialysis assays showed that a striking increase in extracellular excitatory amino acid levels during ischemia was significantly inhibited by pretreatment with citalopram and clomipramine, However, citalopram (4() mg/kg i,p.) did not alter the extracellular amino acid concentrations in normal gerbils, Thus, serotonin reuptake inhibitors have a protective effect against ischemic neuronal damage. Furthermore, the present result suggests that the protective effect is mediated through prevention of tile accumulation of extracellular excitatory amino acids during and after ischemia.
INTRODUCTION Cerebral ischemia induces variable brain damage according to its degree and duration '~. Even a brief period of cerebral ischemia can injure neurons in the brain ~'*. Hippocampal CAI pyramidal neurons are selectively vulnerable to ischemia, and ischemia of 3 or 5 rain duration causes severe neuronal injury in the hippocampal CAI subfield 3'22'24. Ischemia induces various changes in the brain, such as ATP depletion due to deprivation of glucose and oxygen, lactate accumulation and acidosis, an abnormal release of neurotransmitters and calcium influx 3,4,tf'.ts,3t. Particularly, the abnormal release of excitatory amino acids such as glutamate and aspartate in the vulnerable areas is suggested to play an important role in the ischemic neuronal death 1:,'7. However, the role of serotonin (5-hydroxytryptamine; 5-HT) in ischemic neuronal damage is not fully understood. Naftidrofuryl, a 5-HT 2 antagonist, is reported to prevent destruction of hippocampal CA1 pyramidal cells following 5 rain of ischemia in gerbils ~4.
On the other hand, tryptophan, a 5-HT precurser, improves behavioral changes after ischemia in rats ~. In addition Maura et al. reported, using cerebellar slices of rats, that the, Ca" +-dependent release of glutamate is potently inhibited by 5-HT in a concentration-dependent way 2¢'. Therefore, we examined the effects of selective 5-HT reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage as well as on the amount of extraccllular excitatory amino acids using a microdialysis technique in the gerbil hippocampus. MATERIALS AND M E T H O D S
Animals and drugs Male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka, Japan), aged 9-10 weeks and weighing 60-80 g, were used. They were allowed free access to food and water. Citalopram HCi was provided by Zeria Pharmaceutical Co., Ltd. CIomipramine ltCI was purchased from RBi (Natick, MA). The drugs were dissolved in 0.9% physiological saline and injected intrapcritoneally (i.p,) 30 min before ischemia, Experiment !, Effects of citalopram and clomipramine on neuronal damage bl the hippocampal CA l subfield following ischemia.
Correspomh,nce, N. Nakata, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Fax: (81) (22) 272-5818.
49 The animals were anesthetized with 1.5% halothane in a mixture of 30% oxygen and 70% nitrous oxide. Bilateral common carotid arteries were gently exposed and anesthesia was discontinued. The arteries were occluded with aneurysm clips at the initial sign of recover from anesthesia. Ischemia was induced for 5 min. Body and temporalis muscle temperatures were monitored and maintained at more than 37°C during ischemia and for 30 rain after ischemia using a heating pad with thermostat. Four days after ischemia, the animals were killed and the brains were removed and immersed in 10% formalin solution until they were embedded in paraffin. Morphological changes in the hippocampus were examined using hematoxylin-eosin-stained sections. Neuronal damage to the hippocampal CA1 subfield was semiquantitatively graded using a 0-3 rating system with 0: normal, 1: a few neurons damaged, 2: many neurons damaged, and 3: majority of neurons damaged.
Experiment 2. Effects of citalopram and clomipramine on extracelhdar amino acid concentrations in the hippocampal CAI subfield during and after ischemia Implantation of microdialysis probes (1 mm dialysis membrane, 0.22 mm o.d., mol. wt. cut-off = 50,000, Eicom, Japan) was performed under anesthesia with 1.5% haiothane in a mixture of 30% oxygen and 70% nitrous oxide. The probe was implanted from the point 1.0 mm posterior, 2.0 mm lateral and 1.7 mm ventral to the bregma into the CAI region of the hippocampus according to the atlas of Thessen and Yahk "~4. After implantation of the probe, the animals were maintained under light anesthesia (0.5-0.75% halothane in the same O 2 / N 2 0 mixture) for more than 1 h as a stabilization period before the reduction of ischemia. Bilateral carotid arteries were occluded for 5 min under light anesthesia. The probes were perfused with physiological saline at a flow rate of I #,l/min. Dialysate samples were obtained at 10-min ( = 10 /zl of dialysate) intervals and collected in sampling tubes in an ice bath. Amino acid concentrations were measured according to the method of Benveniste et al. s with minor modifications. In brief, the analysis of amino acid concentrations in the dialysates were performed using a high performance liquid chromatography system (HPLC; Eicom, Japan). The dialysate (10 #1) was mixed with 10 #,i of O-phthaldialdehyde. After a 2 rain reaction time, 10 #l of the sample was injected into the ltPLC system with ECD detector. Eicompak MA-5ODS (diameter 4.6x 150 ram, Eicom, Japan)was used for derivatives of amino acids, and 30% methanol in phosphate buffer (0.1 M, pH - 6.0) was used for linear gradient solution. Using these relative response factors, concentrations of amino acids in dialysates were calculated from peak areas in individual chromatograms. The position of microdialysis probe was determined using hematoxylin-eosin-stained sections.
TABLE I
Neuronal damage to the hippocampal CAI subfield 4 days after 5 rain of ischemia Drugs were administered i.p. 30 min before ischemic challenge. The values represent means + S.D.
Drug
Dose
n
Score of damage to CA 1
Sham Saline Citalopram Clomipramine
40 mg/kg 20 mg/kg
6 7 8 5
0 +0 2.71 :t: 0.49 ** 0.88 _+1.36 ## 0.80_+1.30 #
** P < 0.01 compared to sham-operated group (Mann-Whitney U-test). *" ¢* P < 0.05, 0.01 compared to saline-treated group (Mann-Whitney U-test).
protective effect, and the scores were significantly decreased compared to saline-treated animals (Table I).
Experiment 2 The changes in amino acid concentrations of the dialysate are shown in Figs. 1-3. The rate of increase compared to preischemia values are indicated. Amino acid concentrations in the dialysates signifi, eantly increased during 5 rain of ischemia compared to sham-operated animals. During a 5 min period of ischemia, we observed a 10.5-fold increase in aspartate, a 9.3-fold increase in glutamate, a 1.2-fold increase in glutamine, a 1.8-fold increase in glycine, a 3.9-fold increase in taurine, a 1.6-fold increase in alanine, and a
Extraoellular amino acid concentrations %~ehange 2000] 18004
1400"
Experiment 3. Effects of citaiopram on extracellular amino acid concentrations in the hippocampal CA l subfield in normal gerbils. Microdialysis probes were implanted as described above. Under light anesthesia (0.5-0.75% halothane in 0 2/N20), citalopram was administered i.p. to normal gerbils.
1200"
Statistics Statistcal significance was analyzed using the Mann-Whitney U-test. Data were represented as means + S.D.
400
Experiment 1 No morphological changes were observed in shamoperated gerbils. In saline-treated animals subjected to 5 min of ischemia, almost all CA1 neurons were lost and the damage score was 2.7 ± 0.2. Animals treated with citalopram and clomipramine showed a marked
Sham Saline Citalopram Clomipramlne
1000" 800" 600 t
200" 0'--
RESULTS
• • • []
1600"
Aspartate
Glutamate
Ill
Glutamine
Fig. 1. Changes in aspartate, glutamate and glutamine extracellular concentrations in dialysate during a 10-min period including 5 rain of ischemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemia challenge. Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before onset of the ischemic insult. Drugs were administered i.p. 30 min before the ischemic challenge. The columns represent the means+S.D, from 4-6 animals. *, **: P < 0.05, 0.01 vs. sham-operated, #, # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
50 Extrecellular amino acid concentrations
Extracellular GABA concentration
% of change
% of change
600 "1
8000 • • • []
500"
,oo-i
=t
Sham Saline Citalopram Clomipramine
7000
5000
300"
4000 a
,ool
T
6000
i i " .:
i_'.
• • • []
Sham Saline Citalopram Clomipramine
3000
T.
2000 1001000
Glycine
Taurine
Alanlne
Fig. 2. Changes in glycine, taurine and alanine extracellular concentrations in dialysate during a 10-min period including 5 rain of i~chemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemic challenge. Amino acid concentrations in each dialysate were calculated as percentage o| the mean concentration of the dialysate collected before onset of the ischemic insult. Drugs were administered i.p. 30 rain before the ischemic challenge. The columns represent the means+S.D, from 4-6 animals, *, *°: P < 0.05, 0.01 vs. sham-operated. #. # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
44.9-fold increase in gamma-aminobutyric acid (GABA). Pretreatment with citalopram and clomipramine prevented the accumulation of amino acids during ischemia except for aspartate in the citalopram and ciomipramine groups and glutamine in the clomipramine group. In our preliminary experiments, we found no difference in the amino acid concentrations when we used saline as a perfusate instead of Ringer solution.
0 GABA Fig. 3. Changes in GABA extracellular concentrations in dialysate during a 10-rain period including 5 min of ischemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemic challenge. Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before onset of the ischemic insult, Drugs were administered i.p. 3(1 rain before the ischemic challenge. The columns represent the means :t: S.D. from 4-6 animals. *, **: P < 0.05, 0.01 vs sham-operated, #, # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
The location of dialysis probes was situated through hippocampal CAI subfield as confirmed by histology (data not shown).
Experiment 3 Citalopram did not alter the extracellular concentrations ot amino acids in the hippoeampal CAI subfield in normal gerbils (Table I1).
TABLE !! Ef]'ect of citalopram on ettmcelluh~r concentrations of atnino acids in hippocan~pai CA ! subfieM of the. nornml gerbil Dialysates samples were obtained at 10-rain intervals and sample collection started immediately after citalopram administration, Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before drug administration, The values represent means 5: S.D. from 5 animals. Amino acM
Drug
Aspartate
Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram
Glutamate Glutamine Glycine Taurine Alanine GABA
Time aper administration (ram) O- lO
!0 - 20
20- 30
3 0 - 40
103,0+ 4.1 I(X),O+ 0.0 t12,4± 25.2 !!2,65:38.5 1042:1:12,4 98,0+ 2.0 108,2 5:13,7 90,2-t- 17,6 88,4+ 10.5 92,6+ 5.7 i!5,2+ 37,7 90,4+ 7.4 127,0 5:158.6 114,0+ 31.3
98,25: 6,5 I(X).O5: 0.0 85,25:26,6 106,45:il,2 100,05:12.2 91,0_+ 9,4 97.8_+ 12,4 84,3_+ 7.4 79.6_+ 13,9 78,6_+ 6.7 97.0-+ 14.4 82,0:t- il.5 187.2 5:288.4 100,05: 0.0
101.2+ 29,7 100.0 ± 0.0 85,2-t- 23.0 132.05:15,9 98.2+ 17.7 89.6+ 13.2 108.4 :t: 33,2 98.6_+ 48.7 75.0+ 14.6 76,8+ 3.7 105.0+ 35,4 78,0-+ 7.8 137.6 + 183,3 100.0_+ 0.0
119,25:33,3 100.0 5: 0.0 90,0+ 31,0 126.8_+ 34,6 97.2_+ 15.1 85.4+ 16,1 114.0_+ 25,6 98.6_+ 48.7 67.2_+ 10.0 71.6+ 11.3 92.6_+ 32.4 68.4-+ 13.0 136.2_+ 167.1 100.0_+ 0.0
51 DISCUSSION The present result demonstrated that selective 5-HT reuptake inhibitors, citalopram and clomipramine, prevented delayed neuronal death of hippocampal CA1 pyramidal neurons following 5 min ischemia in the gerbil. Furthermore, we also demonstrated that citalopram and clomipramine inhibited the extracellular accumulation of excitatory amino acids during ischemia. Therefore, the protective effects of citalopram and clomipramine against ischemic neuronal damage are suggested to be mediated through inhibition of a massive release of excitatory amino acids during ischemia. It is generally accepted that the elevated excitatory amino acid concentrations during ischemia trigger the chain of reactions that lead to neuronal death t2'27. Previous reports have shown a massive increase in extracellular amino acid levels during ischemia. Benveniste et al. s reported an 8-fold elevation in glutamate, a 3-fold increase in aspartate, and a 2.6-fold increase in taurine levels during 10 rain of ischemia in the rat hippocampus. Subsequent reports showed similar increases in extracellular amino acid levels using different ischemia models t't°'2°'28'2'~.We also observed a 3.6-fold increase in extracellular glutamate concentration during 3 rain of ischemia in the gerbil hippocampus 3°. In the present study, we observed a 9.3-fold increase in the glutamate levels during 5 min of ischemia. Our observation in these two experiments are, thus, in accordance with previous reports. The elevated excitatory amino acid concentration activates receptors. Particularly, activation of N-methyl-D-aspartate (NMDA) receptors induces calcium influx and activation of second-messenger systems such as protein kinase C (PKC) 2t, both of which are considered to play important roles in the development of ischemic neuronal death 23. In fact, NMDA-receptor antagonists, such as MK-801, 3-(-)-2-carboxylpiperazin-4-yl propyl-l-phosphonate (CPP) and 2-amino-7phosphonoheptanoate (AP-7), and PKC inhibitors, such as staurosporine and K-252a, prevent ischemic neuronal damage 7's't5'l'~'35. Therefore, reductions in excitatory amino acid levels during ischemia can protect from the chain of reaction leading to neuronal death. Citalopram and clomipramine inhibited the increase in the extracellular amino acid levels during ischemia. The mechanism of inhibition may be suppression of calcium-evoked glutamate release by serotonin because such a phenomenon is reported by Maura et al. using slices of rat cerebellum 26. It may be noteworthy that citalopram never altered extracellular amino acid concentrations in normal gerbil brain. It is well known that GABA modulates glutamater-
gic activity 33. A previous report has shown that there was a 43-52-fold increase in extracellular GABA level during ischemia in the rat striatum and thalamus using a 2-vessel occlusion combined systemic hypotension model ~7. In the present study, there was a 44.9-fold increase in extracellular concentration of GABA during and after 5 min ischemia. However, pretreatment with citalopram and clomipramine resulted in a significant decrease of GABA levels compared with the saline-treated gerbil. Therefore, it seems probable that the serotonin reuptake inhibitors, citalopram and clomipramine, may not act on the GABAergic nervous system to protect neuronal damage induced by 5 min ischemia. Role of the serotonin system in ischemic neuronal damage is not fully elucidated at present. Fujikura et al. and Krieglstein et al. reported a protective effect of a 5-HT 2 antagonist, naftidrofuryl, on hippocampal neuronal damage in the rat and gerbil 14,25. However, the hippocampal CA1 subfield has few 5 - H T 2 receptors although the neocortex has a dense 5-HT 2 receptor in the rat 32. In this context, the mechanism of protection with naftidrofuryl was suggested to be the inhibition of transsynaptic glutamatergic relays that come from the neocortex ~4. On the other hand, Bielenberg et al. reported that a 5-HTtA agonist, ipsapirone, has a neuroprotective effect in a focal ischemia model in the rat t'. The 5-HTtA receptor is negatively linked to forskolinstimulated adenylate cyclase and is also linked to K ÷ channels as is the adenosine A t receptor t'. An adenosine uptake inhibitor, propentofylline, protects against CA1 neuronal death in the gerbil t3 and reduces extracellular glutamate levels during ischemia in the rat 2. Therefore, the protective effect of citalopram and clomipramine are suggested to be similar to that of propentofylline. In conclusion, we showed 5-HT reuptake inhibitors, citalopram and ciomipramine have a protective effect against ischemic neuronal damage in the gerbil by reducing the extracellular excitatory amino acid levels during ischemia. The present results may warrant clinical application of 5-HT reuptake inhibitors to ischemic cerebrovascular disorders. REFERENCES 1 Andine, P., Orwar, O., Jacobson, I., Sandberg, M. and Hagberg, H., Changes in extracellular amino acids and spontaneous neuronal acivity during ischemia and extended reflow in the CA1 of the rat hippocampus, J. Neurochem., 57 (1991) 222-229. 2 Andine, P., Rudolphi, K.A., Fredholm, B.B. and Hagberg, H., Effect of propentof-ylline (HWA 285) on extracellular purines and excitatory amino acids on CAI of rat hippocampus during transient ischaemia, Br. J. PharmacoL, 100 (1990) 814-818. 3 Araki, T., Kato, H. and Kogure, K., Selective neuronal vulnerabil-
52 ity following transient cerebral ischemia in the gerbil: distribution and time course, Acta Neurol. Scand., 80 (19891 548-553. 4 Araki, T., Kato, H. and Kogure, K., Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil, Brain Res., 528 (19901 114-122. 5 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate ~ rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (19841 1369-1374. 6 Bielenberg, G.W. and Burkhardt. M., 5-Hydroxytryptamine~A agonists. A new therapeutic principle for stroke treatment, Stroke, 21 Suppl, 4 (19901 161-163. 7 Boast, C.A., Gerhardt, S.C. and Janak, P., Systemic AP7 reduced ischemia brain damage in gerbils. In T.P.D. Hicks, D. Lodge and H. McLennan (Eds.), Excitato,tT Amino Acid Transmission, Alan R. Liss, New York, 1987, pp. 249-252. 8 Boast, C.A., Gerhardt, S.C., Pastor, G., Lehmann, J., Etienne, P.E. and Liebman, J.M., The N-methyI-D-aspartate antagonists CGS 19755 and CPP reduce ischemic brain damage in gerbils, Brain Res., 442 (19881 345-348. 9 Brierley, J.B., Cerebral hypoxia. In W. Blackwood, (Eds.), Greenfield~ Neuropatholo~,~.', Edward Arnold, London, 1976. pp.41-85. 10 Butcher,S.P., Bullock. R., Graham, D.I. and Mccuiloch J., Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion, Stroke, 21 (19901 1727-1733. !1 Carney, J.M., Acute tryptophan pretreatment protects against behavioral changes caused by cerebral isehemia, Neurosci. Lett., 66 (19861 127-130. 12 Choi, D.W., Methods for antagonizing glutamate neurotoxicity, Cerebmvasc. Brain Metah. Rt'v., 2 (1990) 105-147. 13 DeL¢o, J., Toth, L., Schubert, P., Rudolphi. K. and Kreutzberg, G., Ischemia-induced neuronal cell death, calcium accumulation, and glial response in the hippocampus of the mongolian gerbil and protection by propentofylline (HWA 285), J. ('en,h. FIh,~d Flow Metab.. 7 (1987) 745-751. 14 Fujikura, It., Kato, !t., Nakano, S. and Kogur¢, K., A scrotonin $2 antagonist (naftidrofuryl) exhibited a marked protective effect on ischemic brain damage in the gerbil (abstract), J. Cereh. BIor~d bYow Metah., 9 Suppl. I (1989) SI83. 15 Gill, R., Foster, A,C. and Woodruff, G.N., Systemic administration of MK 801 protects against ischemia.induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987)3343-3349. 16 GIobus, M.Y.-T., Busto, R., Dietrich, W.D., Martinez E., Valdes, i. and Ginsberg, M.D., Effect of ischemia on the in vivo release of striatal dopamin¢, glutamate, and ~,-aminobutric acid studied by intracer¢bral microdialy,~is, J. Neurochem., 51 (19881 14551464. 17 GIohus, M.Y.-T., Ginsberg, M.D. and Busto, R., Excitotoxic index--a biochemical marker of selective vulnerability, Neurosci. Lett,, 127 (19911 39-42. 18 Graham, S., Shiraishi, K., Panter, S.S., Simon, R.P, ad Faden, A.S.I., Changes in extracellular amino acid neurotransmitter produced by focal cerebral ischemia, Neurosci. Lett., 110 (19901 124-130. 19 Hara, H., Onodera, H., Yashidomi, M., Matsuda, Y. and Kogure, K., Staurosporine, a nevel protein kinase C inhibitor, prevents
postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab., 10 (19901 646-653. 20 Hillered, L., Hallstrom, A., Segersvard, S., Persson, L. and Ungerstedt, U., Dynamic extracellular metabolites in the striatum after middle cerebral artery occlusion in the rat monitored by intracerebral microdialysis, J. Cereb. Blood Flow Metab., 9 (19891 607-616. 21 Jacewicz, M., Kiessling, M. and Pulsenelli, W.A., Selective gene expression in focal cerebral ischemia, J. Cereb. Blood Flow Metab., 9 (19891 607-616. 22 Kato, H., Liu, Y., Araki, T. and Kogure, K., Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects, Brain Res., 553 (1991) 238-242. 23 Kawagoe, J., Hara, H. and Kogure, K., Distribution of protein kinase C in the hippocampus of the gerbil and rat: Autoradiographic analysis by [3H]phorbol 12,13-dibutyrate, J. Pharm. PharmacoL, 43 (1991) 758-761. 24 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 ¢19821 57-69. 25 Krieglstein, J., Sauer, D., Nuglisch, J., Rossberg, C., Beck, T., Bielenberg, G.W., Mennel, H.-D., Naftidrofuryl protects neurons against ischemic damage, Ear. NeuroL, 29 (1989) 224-228. 26 Maura, G., Roccatagliata, E., Ulivi, M. and Raiteri, M., Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT I and 5-HT 2 receptors, Ear. J. Pharmacol., 145 (19881 31-38. 27 Meldrum, B., Protection against ischemic neuronal damage by drugs acting on excitatoxy neurotransmission, Cerebrot'asc. Brain Metab. Rev., 2 (19901 27-57. 28 Mitani, A., Imon, H., lga, K., Kubo, H. and Kataoka, K., Gerbil hippocampal extracellular glutamate and neuronal activity after transient ischemia, Brain Res. BulL, 25 (19901 319-324. 29 Mitani, A., Kubo, !t., lga, K., imon, !t., Kadoya, F. and Kataoka, K.. A new enzymatic cycling technique for glutamate determination in brain microdialysatcs, J. Neurochem., 54 (199(l)7(19-71 I. 30 Nakata, N., Kato, H,, Lit=, Y, and Kogure, K., Effects of pretreatment with sublethal ischemia on the extraccllular glutamate conccntrations during secondary ischemia in the gerbil hippocampus evaluated with intreccrehral microdialysis, NeuroscL Lett., 138 ( 19921 86-88. 31 Nowicki, J,.P., AssumeI-Lurdin, C,, Duvcrger, D, and MacKenzie, E.T,, Temporal evolution of regional energy methabolism following focal cerebral ischemia in the rat, J. Cereb. Blood Flow Metab., 8 (19881 462-473. 32 Pazos, A., Cortes, R. and Palacios, M,J., Quantitative autoradiographic mapping of serotonin receptors in the rat brain. !!, seronotin-2 receptors, Brain Res,, 346 (19851 231-249, 33 Roberts, E., Chase, T.N, and Tower, D,B., GABA in Nen'ous System Function, Raven, New York, 1976, 34 Thiesscn, D, and Yahr, P,, The Gerbil in Behavioral hwestigations, Mechanisms of Territorialio' and Olfactory Comnumication, University of Texas Press, Texas, 1977. 35 Yoshidomi, M., Kogure, K,, Hara, H,, Abe, K. and Matsuda, Y., Inhibition of protein kinase activities ameliorates postischemic brain cell damage, J, Cereb, Blood Flow Metab., 9 (19891 S186.
48
BRES 18044
Protective effects of serotonin reuptake inhibitors, citalopram and clomipramine, against hippocampal CA1 neuronal damage following transient ischemia in the gerbil Naoki N a k a t a , H i r o y u k i K a t o a n d Kyuya K o g u r e Department of Neurology, hastitute of Brain Diseases, Tohoku Unit'ersity School of Medicine, Sendai (Japan) (Accepted 7 April 1992)
Ke.v words: Cerebral ischemia; Serotonin; Citalopram; Clomipramine; Glutamate; Excitatory amino acid; Microdialysis; Gerbil
To clarify the role of serotonin in cerebral ischemia, we examined the effects of selective serotonin reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage in the gerbil. Pretreatment with citalopram (4() mg/kg i.p.) and clomipramine (20 mg/kg i.p.) protected against neuronal destruction of hippocampal CAI pyramidal cells following 5 rain of forebrain ischemia. Furthermore, microdialysis assays showed that a striking increase in extracellular excitatory amino acid levels during ischemia was significantly inhibited by pretreatment with citalopram and clomipramine, However, citalopram (4() mg/kg i,p.) did not alter the extracellular amino acid concentrations in normal gerbils, Thus, serotonin reuptake inhibitors have a protective effect against ischemic neuronal damage. Furthermore, the present result suggests that the protective effect is mediated through prevention of tile accumulation of extracellular excitatory amino acids during and after ischemia.
INTRODUCTION Cerebral ischemia induces variable brain damage according to its degree and duration '~. Even a brief period of cerebral ischemia can injure neurons in the brain ~'*. Hippocampal CAI pyramidal neurons are selectively vulnerable to ischemia, and ischemia of 3 or 5 rain duration causes severe neuronal injury in the hippocampal CAI subfield 3'22'24. Ischemia induces various changes in the brain, such as ATP depletion due to deprivation of glucose and oxygen, lactate accumulation and acidosis, an abnormal release of neurotransmitters and calcium influx 3,4,tf'.ts,3t. Particularly, the abnormal release of excitatory amino acids such as glutamate and aspartate in the vulnerable areas is suggested to play an important role in the ischemic neuronal death 1:,'7. However, the role of serotonin (5-hydroxytryptamine; 5-HT) in ischemic neuronal damage is not fully understood. Naftidrofuryl, a 5-HT 2 antagonist, is reported to prevent destruction of hippocampal CA1 pyramidal cells following 5 rain of ischemia in gerbils ~4.
On the other hand, tryptophan, a 5-HT precurser, improves behavioral changes after ischemia in rats ~. In addition Maura et al. reported, using cerebellar slices of rats, that the, Ca" +-dependent release of glutamate is potently inhibited by 5-HT in a concentration-dependent way 2¢'. Therefore, we examined the effects of selective 5-HT reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage as well as on the amount of extraccllular excitatory amino acids using a microdialysis technique in the gerbil hippocampus. MATERIALS AND M E T H O D S
Animals and drugs Male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka, Japan), aged 9-10 weeks and weighing 60-80 g, were used. They were allowed free access to food and water. Citalopram HCi was provided by Zeria Pharmaceutical Co., Ltd. CIomipramine ltCI was purchased from RBi (Natick, MA). The drugs were dissolved in 0.9% physiological saline and injected intrapcritoneally (i.p,) 30 min before ischemia, Experiment !, Effects of citalopram and clomipramine on neuronal damage bl the hippocampal CA l subfield following ischemia.
Correspomh,nce, N. Nakata, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Fax: (81) (22) 272-5818.
49 The animals were anesthetized with 1.5% halothane in a mixture of 30% oxygen and 70% nitrous oxide. Bilateral common carotid arteries were gently exposed and anesthesia was discontinued. The arteries were occluded with aneurysm clips at the initial sign of recover from anesthesia. Ischemia was induced for 5 min. Body and temporalis muscle temperatures were monitored and maintained at more than 37°C during ischemia and for 30 rain after ischemia using a heating pad with thermostat. Four days after ischemia, the animals were killed and the brains were removed and immersed in 10% formalin solution until they were embedded in paraffin. Morphological changes in the hippocampus were examined using hematoxylin-eosin-stained sections. Neuronal damage to the hippocampal CA1 subfield was semiquantitatively graded using a 0-3 rating system with 0: normal, 1: a few neurons damaged, 2: many neurons damaged, and 3: majority of neurons damaged.
Experiment 2. Effects of citalopram and clomipramine on extracelhdar amino acid concentrations in the hippocampal CAI subfield during and after ischemia Implantation of microdialysis probes (1 mm dialysis membrane, 0.22 mm o.d., mol. wt. cut-off = 50,000, Eicom, Japan) was performed under anesthesia with 1.5% haiothane in a mixture of 30% oxygen and 70% nitrous oxide. The probe was implanted from the point 1.0 mm posterior, 2.0 mm lateral and 1.7 mm ventral to the bregma into the CAI region of the hippocampus according to the atlas of Thessen and Yahk "~4. After implantation of the probe, the animals were maintained under light anesthesia (0.5-0.75% halothane in the same O 2 / N 2 0 mixture) for more than 1 h as a stabilization period before the reduction of ischemia. Bilateral carotid arteries were occluded for 5 min under light anesthesia. The probes were perfused with physiological saline at a flow rate of I #,l/min. Dialysate samples were obtained at 10-min ( = 10 /zl of dialysate) intervals and collected in sampling tubes in an ice bath. Amino acid concentrations were measured according to the method of Benveniste et al. s with minor modifications. In brief, the analysis of amino acid concentrations in the dialysates were performed using a high performance liquid chromatography system (HPLC; Eicom, Japan). The dialysate (10 #1) was mixed with 10 #,i of O-phthaldialdehyde. After a 2 rain reaction time, 10 #l of the sample was injected into the ltPLC system with ECD detector. Eicompak MA-5ODS (diameter 4.6x 150 ram, Eicom, Japan)was used for derivatives of amino acids, and 30% methanol in phosphate buffer (0.1 M, pH - 6.0) was used for linear gradient solution. Using these relative response factors, concentrations of amino acids in dialysates were calculated from peak areas in individual chromatograms. The position of microdialysis probe was determined using hematoxylin-eosin-stained sections.
TABLE I
Neuronal damage to the hippocampal CAI subfield 4 days after 5 rain of ischemia Drugs were administered i.p. 30 min before ischemic challenge. The values represent means + S.D.
Drug
Dose
n
Score of damage to CA 1
Sham Saline Citalopram Clomipramine
40 mg/kg 20 mg/kg
6 7 8 5
0 +0 2.71 :t: 0.49 ** 0.88 _+1.36 ## 0.80_+1.30 #
** P < 0.01 compared to sham-operated group (Mann-Whitney U-test). *" ¢* P < 0.05, 0.01 compared to saline-treated group (Mann-Whitney U-test).
protective effect, and the scores were significantly decreased compared to saline-treated animals (Table I).
Experiment 2 The changes in amino acid concentrations of the dialysate are shown in Figs. 1-3. The rate of increase compared to preischemia values are indicated. Amino acid concentrations in the dialysates signifi, eantly increased during 5 rain of ischemia compared to sham-operated animals. During a 5 min period of ischemia, we observed a 10.5-fold increase in aspartate, a 9.3-fold increase in glutamate, a 1.2-fold increase in glutamine, a 1.8-fold increase in glycine, a 3.9-fold increase in taurine, a 1.6-fold increase in alanine, and a
Extraoellular amino acid concentrations %~ehange 2000] 18004
1400"
Experiment 3. Effects of citaiopram on extracellular amino acid concentrations in the hippocampal CA l subfield in normal gerbils. Microdialysis probes were implanted as described above. Under light anesthesia (0.5-0.75% halothane in 0 2/N20), citalopram was administered i.p. to normal gerbils.
1200"
Statistics Statistcal significance was analyzed using the Mann-Whitney U-test. Data were represented as means + S.D.
400
Experiment 1 No morphological changes were observed in shamoperated gerbils. In saline-treated animals subjected to 5 min of ischemia, almost all CA1 neurons were lost and the damage score was 2.7 ± 0.2. Animals treated with citalopram and clomipramine showed a marked
Sham Saline Citalopram Clomipramlne
1000" 800" 600 t
200" 0'--
RESULTS
• • • []
1600"
Aspartate
Glutamate
Ill
Glutamine
Fig. 1. Changes in aspartate, glutamate and glutamine extracellular concentrations in dialysate during a 10-min period including 5 rain of ischemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemia challenge. Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before onset of the ischemic insult. Drugs were administered i.p. 30 min before the ischemic challenge. The columns represent the means+S.D, from 4-6 animals. *, **: P < 0.05, 0.01 vs. sham-operated, #, # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
50 Extrecellular amino acid concentrations
Extracellular GABA concentration
% of change
% of change
600 "1
8000 • • • []
500"
,oo-i
=t
Sham Saline Citalopram Clomipramine
7000
5000
300"
4000 a
,ool
T
6000
i i " .:
i_'.
• • • []
Sham Saline Citalopram Clomipramine
3000
T.
2000 1001000
Glycine
Taurine
Alanlne
Fig. 2. Changes in glycine, taurine and alanine extracellular concentrations in dialysate during a 10-min period including 5 rain of i~chemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemic challenge. Amino acid concentrations in each dialysate were calculated as percentage o| the mean concentration of the dialysate collected before onset of the ischemic insult. Drugs were administered i.p. 30 rain before the ischemic challenge. The columns represent the means+S.D, from 4-6 animals, *, *°: P < 0.05, 0.01 vs. sham-operated. #. # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
44.9-fold increase in gamma-aminobutyric acid (GABA). Pretreatment with citalopram and clomipramine prevented the accumulation of amino acids during ischemia except for aspartate in the citalopram and ciomipramine groups and glutamine in the clomipramine group. In our preliminary experiments, we found no difference in the amino acid concentrations when we used saline as a perfusate instead of Ringer solution.
0 GABA Fig. 3. Changes in GABA extracellular concentrations in dialysate during a 10-rain period including 5 min of ischemia in the gerbil hippocampus. Collection of dialysate started immediately after induction of ischemic challenge. Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before onset of the ischemic insult, Drugs were administered i.p. 3(1 rain before the ischemic challenge. The columns represent the means :t: S.D. from 4-6 animals. *, **: P < 0.05, 0.01 vs sham-operated, #, # # : P < 0.05, 0.01 vs. saline control, Mann-Whitney U-test.
The location of dialysis probes was situated through hippocampal CAI subfield as confirmed by histology (data not shown).
Experiment 3 Citalopram did not alter the extracellular concentrations ot amino acids in the hippoeampal CAI subfield in normal gerbils (Table I1).
TABLE !! Ef]'ect of citalopram on ettmcelluh~r concentrations of atnino acids in hippocan~pai CA ! subfieM of the. nornml gerbil Dialysates samples were obtained at 10-rain intervals and sample collection started immediately after citalopram administration, Amino acid concentrations in each dialysate were calculated as percentage of the mean concentration of the dialysate collected before drug administration, The values represent means 5: S.D. from 5 animals. Amino acM
Drug
Aspartate
Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram Sham Citalopram
Glutamate Glutamine Glycine Taurine Alanine GABA
Time aper administration (ram) O- lO
!0 - 20
20- 30
3 0 - 40
103,0+ 4.1 I(X),O+ 0.0 t12,4± 25.2 !!2,65:38.5 1042:1:12,4 98,0+ 2.0 108,2 5:13,7 90,2-t- 17,6 88,4+ 10.5 92,6+ 5.7 i!5,2+ 37,7 90,4+ 7.4 127,0 5:158.6 114,0+ 31.3
98,25: 6,5 I(X).O5: 0.0 85,25:26,6 106,45:il,2 100,05:12.2 91,0_+ 9,4 97.8_+ 12,4 84,3_+ 7.4 79.6_+ 13,9 78,6_+ 6.7 97.0-+ 14.4 82,0:t- il.5 187.2 5:288.4 100,05: 0.0
101.2+ 29,7 100.0 ± 0.0 85,2-t- 23.0 132.05:15,9 98.2+ 17.7 89.6+ 13.2 108.4 :t: 33,2 98.6_+ 48.7 75.0+ 14.6 76,8+ 3.7 105.0+ 35,4 78,0-+ 7.8 137.6 + 183,3 100.0_+ 0.0
119,25:33,3 100.0 5: 0.0 90,0+ 31,0 126.8_+ 34,6 97.2_+ 15.1 85.4+ 16,1 114.0_+ 25,6 98.6_+ 48.7 67.2_+ 10.0 71.6+ 11.3 92.6_+ 32.4 68.4-+ 13.0 136.2_+ 167.1 100.0_+ 0.0
51 DISCUSSION The present result demonstrated that selective 5-HT reuptake inhibitors, citalopram and clomipramine, prevented delayed neuronal death of hippocampal CA1 pyramidal neurons following 5 min ischemia in the gerbil. Furthermore, we also demonstrated that citalopram and clomipramine inhibited the extracellular accumulation of excitatory amino acids during ischemia. Therefore, the protective effects of citalopram and clomipramine against ischemic neuronal damage are suggested to be mediated through inhibition of a massive release of excitatory amino acids during ischemia. It is generally accepted that the elevated excitatory amino acid concentrations during ischemia trigger the chain of reactions that lead to neuronal death t2'27. Previous reports have shown a massive increase in extracellular amino acid levels during ischemia. Benveniste et al. s reported an 8-fold elevation in glutamate, a 3-fold increase in aspartate, and a 2.6-fold increase in taurine levels during 10 rain of ischemia in the rat hippocampus. Subsequent reports showed similar increases in extracellular amino acid levels using different ischemia models t't°'2°'28'2'~.We also observed a 3.6-fold increase in extracellular glutamate concentration during 3 rain of ischemia in the gerbil hippocampus 3°. In the present study, we observed a 9.3-fold increase in the glutamate levels during 5 min of ischemia. Our observation in these two experiments are, thus, in accordance with previous reports. The elevated excitatory amino acid concentration activates receptors. Particularly, activation of N-methyl-D-aspartate (NMDA) receptors induces calcium influx and activation of second-messenger systems such as protein kinase C (PKC) 2t, both of which are considered to play important roles in the development of ischemic neuronal death 23. In fact, NMDA-receptor antagonists, such as MK-801, 3-(-)-2-carboxylpiperazin-4-yl propyl-l-phosphonate (CPP) and 2-amino-7phosphonoheptanoate (AP-7), and PKC inhibitors, such as staurosporine and K-252a, prevent ischemic neuronal damage 7's't5'l'~'35. Therefore, reductions in excitatory amino acid levels during ischemia can protect from the chain of reaction leading to neuronal death. Citalopram and clomipramine inhibited the increase in the extracellular amino acid levels during ischemia. The mechanism of inhibition may be suppression of calcium-evoked glutamate release by serotonin because such a phenomenon is reported by Maura et al. using slices of rat cerebellum 26. It may be noteworthy that citalopram never altered extracellular amino acid concentrations in normal gerbil brain. It is well known that GABA modulates glutamater-
gic activity 33. A previous report has shown that there was a 43-52-fold increase in extracellular GABA level during ischemia in the rat striatum and thalamus using a 2-vessel occlusion combined systemic hypotension model ~7. In the present study, there was a 44.9-fold increase in extracellular concentration of GABA during and after 5 min ischemia. However, pretreatment with citalopram and clomipramine resulted in a significant decrease of GABA levels compared with the saline-treated gerbil. Therefore, it seems probable that the serotonin reuptake inhibitors, citalopram and clomipramine, may not act on the GABAergic nervous system to protect neuronal damage induced by 5 min ischemia. Role of the serotonin system in ischemic neuronal damage is not fully elucidated at present. Fujikura et al. and Krieglstein et al. reported a protective effect of a 5-HT 2 antagonist, naftidrofuryl, on hippocampal neuronal damage in the rat and gerbil 14,25. However, the hippocampal CA1 subfield has few 5 - H T 2 receptors although the neocortex has a dense 5-HT 2 receptor in the rat 32. In this context, the mechanism of protection with naftidrofuryl was suggested to be the inhibition of transsynaptic glutamatergic relays that come from the neocortex ~4. On the other hand, Bielenberg et al. reported that a 5-HTtA agonist, ipsapirone, has a neuroprotective effect in a focal ischemia model in the rat t'. The 5-HTtA receptor is negatively linked to forskolinstimulated adenylate cyclase and is also linked to K ÷ channels as is the adenosine A t receptor t'. An adenosine uptake inhibitor, propentofylline, protects against CA1 neuronal death in the gerbil t3 and reduces extracellular glutamate levels during ischemia in the rat 2. Therefore, the protective effect of citalopram and clomipramine are suggested to be similar to that of propentofylline. In conclusion, we showed 5-HT reuptake inhibitors, citalopram and ciomipramine have a protective effect against ischemic neuronal damage in the gerbil by reducing the extracellular excitatory amino acid levels during ischemia. The present results may warrant clinical application of 5-HT reuptake inhibitors to ischemic cerebrovascular disorders. REFERENCES 1 Andine, P., Orwar, O., Jacobson, I., Sandberg, M. and Hagberg, H., Changes in extracellular amino acids and spontaneous neuronal acivity during ischemia and extended reflow in the CA1 of the rat hippocampus, J. Neurochem., 57 (1991) 222-229. 2 Andine, P., Rudolphi, K.A., Fredholm, B.B. and Hagberg, H., Effect of propentof-ylline (HWA 285) on extracellular purines and excitatory amino acids on CAI of rat hippocampus during transient ischaemia, Br. J. PharmacoL, 100 (1990) 814-818. 3 Araki, T., Kato, H. and Kogure, K., Selective neuronal vulnerabil-
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