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N-Methyl-d -aspartate receptor blockade by dizocilpine prevents stress-induced sudden death in cardiomyopathic hamsters
Brain Research 944 (2002) 200–204 www.elsevier.com / locate / bres
Short communication
N -Methyl-D-aspartate receptor blockade by dizocilpine prevents stressinduced sudden death in cardiomyopathic hamsters Nobuya Matsuoka*, Hiroshi Kodama, Hiroyuki Arakawa, Isamu Yamaguchi Basic Research Group, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Accepted 6 February 2002
Abstract We previously demonstrated that repeated cold-immobilization stress exposure had lethal effects in cardiomyopathic Syrian hamsters. To clarify the mechanisms of the sudden death, we investigated the effects of N-methyl-D-aspartate (NMDA) receptor blockade by dizocilpine on the sudden death of cardiomyopathic hamsters. Repeated exposure (5 days) to cold-immobilization stress induced a lethal effect in the cardiomyopathic hamsters in agreement with our previous results. Dizocilpine (0.1–10 mg / kg, i.p.), administered just prior to the stress, for 5 consecutive days markedly prevented the lethal effects of the stress. It was further demonstrated that treatment drug significantly reduced the observed increase in organ weights. These results suggest that NMDA receptors have an important role in stress-induced sudden death in cardiomyopathic hamsters and provide the first evidence for the potential therapeutic value of NMDA antagonists against cardiac sudden death. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Stress Keywords: Stress; Cardiomyopathic hamster; Sudden death; Heart failure; Kidney failure; NMDA receptor; Dizocilpine (MK-801)
Stress has been characterized clinically and experimentally to contribute to the development or exacerbation of cardiovascular dysfunction, and is identified as a risk factor for hypertension, cardiac dysrhythmia and sudden cardiac death [8,16]. Although the precise mechanisms of cardiac sudden death are not fully understood, increasing evidence indicates that sudden death resulting from ventricular fibrillation may be triggered by behavioral and neural factors [13,15]. Various stressors which augment sympathetic neural traffic to the heart reportedly lower the vulnerable period threshold for ventricular fibrillation, resulting in sudden death in laboratory animals as well as in humans [13,14,21]. Cardiomyopathic Syrian hamster (BIO 14.6) is known to develop a genetically determined cardiomyopathy, with progressive development of congestive heart failure, re*Corresponding author. Department of Neuroscience, Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan. Tel.: 181-6-6390-1153; fax: 181-6-6304-5367. E-mail address: nobuya [email protected] (N. Matsuoka). ]
sembling human congestive cardiomyopathies [2,27]. We have recently demonstrated that certain forms of stress accelerate the death of the cardiomyopathic Syrian hamsters and proposed this as a putative animal model of cardiac sudden death [17]. Furthermore, we have shown that sudden death of cardiomyopathic hamsters is prevented by propranolol but not by atropine [17], suggesting an important role of sympathetic nerves in the etiology of stress-induced cardiac sudden death. These results have prompted us to explore the neural mechanisms that convey the stress information leading to excitation of the sympathetic nerves and result in the sudden death of cardiomyopathic hamsters. We have recently found that central mechanisms mediated by dopamine D1 / 5 receptors could be one basis for such an activation of sympathetic drive [1]. A growing body of evidence has implicated that excitatory amino acid neurotransmitters and their receptors, the principal mediators of excitatory neurotransmission within the central nervous system (CNS), have a vital role in regulating CNS-mediated physiological functions like cardiovascular and autonomic functions [9,18]. For example,
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02885-8
N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
N-methyl-D-aspartate (NMDA) receptors, one group of the diverse range of glutamate receptor subtypes, reportedly play a role in regulation of blood pressure, heart rate [12,25] and autonomic discharge [5]. Moreover, recent evidence has suggested that central NMDA receptors may participate in the cascade of stress-induced cardiovascular responses [26]. From these findings, we hypothesized that activation of brain NMDA receptors could be involved in disorders associated with stress. Therefore, the aim of the present study was to clarify the role of NMDA receptors in stress-induced sudden death. In this study, the effects of NMDA receptor blockade by dizocilpine on sudden death were investigated using cardiomyopathic Syrian hamsters subjected to cold-immobilization stress. The animals used were 2-month-old male BIO 14.6 cardiomyopathic hamsters and age-matched F1B healthy hamsters purchased from Canadian Hybrid Farms (Nova Scotia, Canada). Hamsters were housed individually in plastic cages in a temperature-controlled environment (2261 8C) under a 12 h light:dark schedule with lights off at 15:00 h and given unlimited access to Purina mouse chow and tap water. The animals were acclimated to these conditions for 4 weeks before the experiment. All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co., Ltd. All experiments were performed according to the method described previously [17]. Briefly, hamsters of each group were further subdivided into stress and non-stress groups. The stress protocol was carried out on 5 consecutive days and began at 15:00 h. The stressed hamsters, including the healthy control strain, were subjected daily to 1 h periods of supine immobilization at 4 8C. This consisted of immobilizing the animal by extending their four limbs and taping them to the corners of a small board then putting them in a refrigerator for 1 h. The non-stressed healthy and cardiomyopathic hamsters were not immobilized and just left in their housing cages outside the refrigerator for 1 h. Immediately before and after each stress session as well as the next morning after each session, the hamsters were checked for body weight and to see if they were still alive. Thereafter, we checked the hamsters twice daily until 7 days after the last stress session. Hamsters were autopsied after either being found dead or after decapitation on the final day of the experiment (day 12). In the animals that succumbed to sudden death, 10 h was the maximum time that was allowed to elapse between death and autopsy. Autopsy consisted of removal of the organs and absorption of pleural and peritoneal fluid on a pre-weighed gauze pad. Organs and body cavity fluid (BCF) were then weighed quickly. The surviving animals were sacrificed at the end of the experiment, 7 days after the last stress session, and autopsied as above. Dizocilpine, purchased from Research Biochemicals Incorporated (Natik, MA), was dissolved in physiological
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saline just before the tests and given intraperitoneally in a volume of 2 ml / kg just prior to the immobilization stress for 5 consecutive days. All results were expressed as mean6S.E.M. Statistical significance of differences in the changes in organ weights was calculated either by using one-way analysis of variance followed by Dunnett’s multiple comparison post-hoc test for multiple dosed groups, or by unpaired Student’s t-test for two group comparison between non-stressed and stressed animals. Mortality results were analyzed by Fisher’s exact probability test. Cumulative surviving percentages of hamsters during the course of the experiments were analyzed using a generalized Wilcoxon test. Neither healthy hamsters with or without stress, nor non-stressed cardiomyopathic hamsters succumbed to sudden death during the course of the experiment (Fig. 1A). In contrast, five out of seven stressed cardiomyopathic hamsters died and there was a statistically significant difference between the mortality of the two groups (P,0.05 by Fisher’s exact probability test). As shown in Fig. 1B, which represents the cumulative surviving percent of hamsters, three animals among the stressed-cardiomyopathic hamsters succumbed during the 5 days of stress sessions, and another two animals died after the stress termination. The difference between the two groups was statistically significant (P,0.05 by generalized Wilcoxon test). Dizocilpine was evaluated for its effect on the sudden death of stressed cardiomyopathic hamsters. Administration of dizocilpine (0.1–10 mg / kg, i.p.) reduced the mortality seen in the stressed-cardiomyopathic hamsters with a bell-shaped dose–response relationship. Dizocilpine provided the maximal protection at 1 mg / kg, with six out of seven animals surviving at the completion of the experiments. However, this change did not reach statistical significance, probably due to the limited number of animals employed for each experimental group (by Fisher’s exact probability test). On the other hand, the highest dose of dizocilpine (10 mg / kg) lost its protective efficacy and most of the animals in that group succumbed during stress sessions, probably because the dose level produced harmful side-effects like hypothermia during stress session which could aggravate the response by cold-immobilization stress. Six days after the completion of the stress sessions, BCF and organ weights were measured (Fig. 2). The stress caused no detectable changes in healthy animals. However, the BCF, heart, adrenal, liver and kidney weights were significantly (P,0.05 by Student’s t-test) increased in the stressed cardiomyopathic hamsters compared with those of non-stressed cardiomyopathic animals. As shown in Fig. 2, administration of dizocilpine significantly prevented increases in the weights of BCF, adrenal, kidney and liver in the stressed-cardiomyopathic hamsters, with statistically significant (P,0.05) differences in the groups dosed with 1 or 10 mg / kg of
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N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
Fig. 1. (A) Effects of dizocilpine (0.1–10 mg / kg) on sudden death caused by immobilization stress in cardiomyopathic hamsters. Each value represents the percentage of surviving hamsters. Numbers in parentheses indicate the number of animals in each group. C, control non-stressed animals; S, stressed animals. (B) Survival curves of unstressed and stressed cardiomyopathic hamsters receiving dizocilpine treatment. Dizocilpine was administered i.p. just prior to the stress, for 5 consecutive days in the first week. Saline was administered to the vehicle control group.
dizocilpine (Fig. 2). The drug only marginally affected the heart weights. Agreeing with the result of a previous study by this group [17], the present study showed that cold-immobilization stress exerted a lethal effect on cardiomyopathic Syrian hamsters but not on control healthy hamsters, suggesting that the stress had serious and even lethal consequences in cardiovascular dysfunction seen in cardiomyopathic hamsters with a covert heart disease [3,27]. Our previous studies demonstrated that severe arrhythmia
was observed before sudden death in stressed-cardiomyopathic hamsters, implying that acute heart failure and / or lethal arrhythmia might be responsible for the death of the stressed cardiomyopathic hamsters [17]. Supporting this view, heart and adrenal weights were markedly increased in those animals, as confirmed in the present study. It was also supported by results indicating that stress elevated BCF in these animals, an index which is suggestive of the presence of fluid retention in the body cavity commonly seen in animals with congestive heart failure
N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
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Fig. 2. Effects of dizocilpine on organ weights in stressed cardiomyopathic hamsters. Each column and bar represent the mean6S.E.M. C, control non-stressed animals; S, stressed animals. * P,0.05, ** P,0.01; significantly different compared with saline-treated stressed cardiomyopathic hamsters (by one-way analysis of variance followed by Dunnett’s multiple comparison test). * P,0.05, *** P,0.001; significantly different from non-stressed cardiomyopathic group (by unpaired Student’s t-test). [[ P,0.01, [[[ P,0.001; significantly different from non-stressed healthy animals (by unpaired Student’s t-test).
[20]. Given the fact that the stress produced a marked increase not only in kidney weight but also in serum levels of urea nitrogen and creatinine in the cardiomyopathic hamsters [17], this likely indicates a reduction in glomerular filtration rate of the kidney possibly related to a decrease in myocardial performance, indicative of sudden ventricular dysfunction. The major finding of the present study is that the NMDA receptor antagonist, dizocilpine, prevented sudden death induced by cold-immobilization stress in cardiomyopathic hamsters, and also significantly ameliorated the stressinduced increase in organ weights. These results clearly demonstrate an involvement of NMDA receptors in sudden death of stressed-cardiomyopathic hamsters. We have previously suggested that an activation of the sympathetic nerves triggered by stress participates in the sudden death in this animal model, as a consequence of increased incidence of heart failure and / or cardiac arrhythmia [17]. Though the present study does not elucidate the precise mechanisms of NMDA receptor participation in stressinduced sudden death, it favors the speculation that central NMDA receptors could play a role in the mechanisms by which stress information is conveyed from the brain to the sympathetic nerves [8,24]. Accumulating evidence has suggested that the stimulation of excitatory amino acids receptors in brain areas such as the amygdala, hypothalamus or lower brain areas activate sympathetic nerves and could result in the abnormal changes in cardiovascular function, such as the loss in heart rhythm and even myocardial damage [5,10,12,23].
Pharmacological blockade of glutamate receptors at these sites eliminates a variety of centrally-mediated cardiovascular responses [4,22]. These include baroreceptor reflexes and increases in arterial pressure produced by both stimulation of various brain regions as well as peripheral afferent nerves [22]. There have also been numerous findings suggesting that central glutamatergic neurotransmission plays a pivotal role in stress-induced cardiovascular response. For instance, Soltis and DiMicco showed that hypothalamic injections of NMDA and AMPA receptors antagonists reduced tachycardia induced by stress in rats [26], suggesting an involvement of intrahypothalamic NMDA receptors in that response. Therefore, it is conceivable that activation of NMDA receptors in brain regions like the amygdala, hypothalamus and brain stem could contribute to the etiological cause of sudden death of stressed-cardiomyopathic hamsters as a consequence of sympathetic excitation. The present study cannot rule out the possible involvement of peripheral NMDA receptors located at sympathetic nerves or nerve terminals at vasculature in the heart, kidney and adrenal glands in the preventive effect of dizocilpine on sudden death. In the present study, dizocilpine reduced the increase in adrenal weight produced by stress, indicating that the drug could effectively attenuate sympathetic nerve discharge. This view is supported by earlier findings of other studies, suggesting the involvement of NMDA receptors at the spinal level in the control of sympathetic output [5]. Further investigations will, however, be required to characterize the detailed
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N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
mechanisms by which blockade of NMDA receptors by dizocilpine results in the prevention of sudden death in stressed-cardiomyopathic hamsters. We have previously demonstrated, using the same model, that dopamine D1 / 5 receptors could be involved in the sudden death of cardiomyopathic hamsters [1]. Therefore, it would also be fascinating to delineate the relationship of D1-mediated mechanisms and NMDA-mediated ones in regulating stress-induced cardiovascular malfunction. Dopaminergic and glutamatergic transmissions have long been known to interact at multiple levels in the striatum and in midbrain dopaminergic areas (substantia nigra and ventral tegmental area) [7]. The interaction could be involved in stress-response, since NMDA receptors have been known to play as important signals of stressinduced enhancement in dopaminergic neurotransmission in certain brain areas [6,11]. One of the dopamine-glutamate interactions is represented by the reciprocal modulation of each other’s release at synaptic terminals. However, it has been recently shown that an activation of D1 receptors, but not of D2 receptors, potentiates transsynaptic responses mediated by activation of NMDA receptors [19]. This specific mode of D1-NMDA interaction may partly explain the neural mechanisms involved in the stressinduced cardiac sudden death. In conclusion, the present results have shown that the blockade of NMDA receptors by dizocilpine significantly prevented sudden death of cardiomyopathic hamsters induced by the cold-immobilization stress, suggesting that NMDA receptors have an important role in the etiology of stress-induced cardiac sudden death of cardiomyopathic hamsters. These findings provide the first experimental evidence for the potential therapeutic value of NMDA antagonists for cardiac sudden death associated with stress.
References [1] H. Arakawa, H. Kodama, I. Yamaguchi, N. Matsuoka, A dopamine D(1 / 5) receptor antagonist, SCH23390, prevents stress-induced sudden death in cardiomyopathic hamsters, Pharmacol. Biochem. Behav. 66 (2000) 707–712. [2] E. Bajusz, J.R. Baker, C.W. Nixon, F. Homburger, Spontaneous, hereditary myocardial degeneration and congestive heart failure in a strain of syrian hamsters, Ann. NY Acad. Sci. 138 (1966) 213–292. [3] E. Bajusz, A. Lossnitzer, A new disease model of congestive heart failure: studies on its pathogenesis, Trans. NY Acad. Sci. 30 (1968) 939–948. [4] M.K. Bazil, F.J. Gordon, Blockade of parabrachial pressor responses by spinal administration of an N-methyl-D-aspartic acid receptor antagonist, Neuropharmacology 29 (1990) 923–930. [5] M.K. Bazil, F.J. Gordon, Sympathoexcitation from the rostral ventrolateral medulla is mediated by spinal NMDA receptors, Brain Res. Bull. 31 (1993) 273–278. [6] S.L. Castro, M.J. Zigmond, Stress-induced increase in extracellular dopamine in striatum: role of glutamatergic action via N-methyl-Daspartate receptors in substantia nigra, Brain Res. 901 (2001) 47– 54.
[7] C. Cepeda, M.S. Levine, Dopamine and N-methyl-D-aspartate receptor interactions in the neostriatum, Dev. Neurosci. 20 (1998) 1–18. [8] R.A. Galosy, L.K. Clarke, M.R. Vasko, I.L. Crawford, Neurophysiology and neuropharmacology of cardiovascular regulation and stress, Neurosci. Biobehav. Rev. 5 (1981) 137–175. [9] F.J. Gordon, Excitatory amino acid receptors in central cardiovascular regulation, Clin. Exp. Hypertens. 17 (1995) 81–90. [10] Y. Hong, J.L. Henry, Intrathecal administration of non-NMDA receptor agonists increases arterial pressure and heart rate in the rat, Brain Res. 591 (1992) 62–68. [11] H.P. Jedema, B. Moghaddam, Glutamatergic control of dopamine release during stress in the rat prefrontal cortex, J. Neurochem. 63 (1994) 785–788. [12] C.B. Jin, R.W. Rockhold, Effects of paraventricular hypothalamic microinfusions of kainic acid on cardiovascular and renal excretory function in conscious rats, J. Pharmacol. Exp. Ther. 251 (1989) 969–975. [13] T. Kamarck, J.R. Jennings, Biobehavioral factors in sudden cardiac death, Psychol. Bull. 109 (1991) 42–75. [14] B.S. Kolman, R.L. Verrier, B. Lown, Effect of vagus nerve stimulation upon excitability of the canine ventricle. Role of sympathetic– parasympathetic interactions, Am. J. Cardiol. 37 (1976) 1041–1045. [15] B. Lown, Sudden cardiac death: biobehavioral perspective, Circulation 76 (1987) I186–I196. [16] T.A. Manolio, C.D. Furberg, Epidemiology of sudden cardiac death, in: M. Akhtar, R.J. Myerburg, J.N. Ruskin (Eds.), Sudden Cardiac Death: Prevalence, Mechanisms, And Approaches To Diagnosis and Management, Williams and Wilkins, Philadelphia, 1994, pp. 3–20. [17] N. Matsuoka, H. Arakawa, H. Kodama, I. Yamaguchi, Characterization of stress-induced sudden death in cardiomyopathic hamsters, J. Pharmacol. Exp. Ther. 284 (1998) 125–135. [18] R.B. McCall, Role of neurotransmitters in the central regulation of the cardiovascular system, Prog. Drug Res. 35 (1990) 25–84. [19] M. Morari, M. Marti, S. Sbrenna, K. Fuxe, C. Bianchi, L. Beani, Reciprocal dopamine-glutamate modulation of release in the basal ganglia, Neurochem. Int. 33 (1998) 383–397. [20] J.E. Ottenweller, W.N. Tapp, T.S. Chen, B.H. Natelson, Cardiovascular aging in Syrian hamsters: similarities between normal aging and disease, Exp. Aging Res. 13 (1987) 73–84. [21] G.W. Parker, L.H. Michael, M.L. Entman, An animal model to examine the response to environmental stress as a factor in sudden cardiac death, Am. J. Cardiol. 60 (1987) 9J–14J. [22] R.W. Rockhold, Excitatory amino acid receptor antagonists: potential implications for cardiovascular therapy and cocaine intoxication, Med. Hypotheses 35 (1991) 342–348. [23] R.W. Rockhold, C.G. Acuff, B.R. Clower, Excitotoxin-induced myocardial necrosis, Eur. J. Pharmacol. 166 (1989) 571–576. [24] J.E. Skinner, Brain involvement in cardivascular disorders, in: T. Elbert, W. Langosch, A. Steptoe, D. Vaitl (Eds.), Behavioural Medicine in Cardiovascular Disorders, Wiley, New York, 1988, pp. 229–253. [25] R.P. Soltis, J.A. DiMicco, Interaction of hypothalamic GABAA and excitatory amino acid receptors controlling heart rate in rats, Am. J. Physiol. 261 (1991) R427–R433. [26] R.P. Soltis, J.A. DiMicco, Hypothalamic excitatory amino acid receptors mediate stress-induced tachycardia in rats, Am. J. Physiol. 262 (1992) R689–R697. [27] J.E. Strobeck, S.M. Factor, A. Bhan, M. Sole, C.C. Liew, F. Fein, E.H. Sonnenblick, Hereditary and acquired cardiomyopathies in experimental animals: mechanical, biochemical, and structural features, Ann. NY Acad. Sci. 317 (1979) 59–88.
Short communication
N -Methyl-D-aspartate receptor blockade by dizocilpine prevents stressinduced sudden death in cardiomyopathic hamsters Nobuya Matsuoka*, Hiroshi Kodama, Hiroyuki Arakawa, Isamu Yamaguchi Basic Research Group, Tsukuba Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Accepted 6 February 2002
Abstract We previously demonstrated that repeated cold-immobilization stress exposure had lethal effects in cardiomyopathic Syrian hamsters. To clarify the mechanisms of the sudden death, we investigated the effects of N-methyl-D-aspartate (NMDA) receptor blockade by dizocilpine on the sudden death of cardiomyopathic hamsters. Repeated exposure (5 days) to cold-immobilization stress induced a lethal effect in the cardiomyopathic hamsters in agreement with our previous results. Dizocilpine (0.1–10 mg / kg, i.p.), administered just prior to the stress, for 5 consecutive days markedly prevented the lethal effects of the stress. It was further demonstrated that treatment drug significantly reduced the observed increase in organ weights. These results suggest that NMDA receptors have an important role in stress-induced sudden death in cardiomyopathic hamsters and provide the first evidence for the potential therapeutic value of NMDA antagonists against cardiac sudden death. 2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Stress Keywords: Stress; Cardiomyopathic hamster; Sudden death; Heart failure; Kidney failure; NMDA receptor; Dizocilpine (MK-801)
Stress has been characterized clinically and experimentally to contribute to the development or exacerbation of cardiovascular dysfunction, and is identified as a risk factor for hypertension, cardiac dysrhythmia and sudden cardiac death [8,16]. Although the precise mechanisms of cardiac sudden death are not fully understood, increasing evidence indicates that sudden death resulting from ventricular fibrillation may be triggered by behavioral and neural factors [13,15]. Various stressors which augment sympathetic neural traffic to the heart reportedly lower the vulnerable period threshold for ventricular fibrillation, resulting in sudden death in laboratory animals as well as in humans [13,14,21]. Cardiomyopathic Syrian hamster (BIO 14.6) is known to develop a genetically determined cardiomyopathy, with progressive development of congestive heart failure, re*Corresponding author. Department of Neuroscience, Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan. Tel.: 181-6-6390-1153; fax: 181-6-6304-5367. E-mail address: nobuya [email protected] (N. Matsuoka). ]
sembling human congestive cardiomyopathies [2,27]. We have recently demonstrated that certain forms of stress accelerate the death of the cardiomyopathic Syrian hamsters and proposed this as a putative animal model of cardiac sudden death [17]. Furthermore, we have shown that sudden death of cardiomyopathic hamsters is prevented by propranolol but not by atropine [17], suggesting an important role of sympathetic nerves in the etiology of stress-induced cardiac sudden death. These results have prompted us to explore the neural mechanisms that convey the stress information leading to excitation of the sympathetic nerves and result in the sudden death of cardiomyopathic hamsters. We have recently found that central mechanisms mediated by dopamine D1 / 5 receptors could be one basis for such an activation of sympathetic drive [1]. A growing body of evidence has implicated that excitatory amino acid neurotransmitters and their receptors, the principal mediators of excitatory neurotransmission within the central nervous system (CNS), have a vital role in regulating CNS-mediated physiological functions like cardiovascular and autonomic functions [9,18]. For example,
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02885-8
N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
N-methyl-D-aspartate (NMDA) receptors, one group of the diverse range of glutamate receptor subtypes, reportedly play a role in regulation of blood pressure, heart rate [12,25] and autonomic discharge [5]. Moreover, recent evidence has suggested that central NMDA receptors may participate in the cascade of stress-induced cardiovascular responses [26]. From these findings, we hypothesized that activation of brain NMDA receptors could be involved in disorders associated with stress. Therefore, the aim of the present study was to clarify the role of NMDA receptors in stress-induced sudden death. In this study, the effects of NMDA receptor blockade by dizocilpine on sudden death were investigated using cardiomyopathic Syrian hamsters subjected to cold-immobilization stress. The animals used were 2-month-old male BIO 14.6 cardiomyopathic hamsters and age-matched F1B healthy hamsters purchased from Canadian Hybrid Farms (Nova Scotia, Canada). Hamsters were housed individually in plastic cages in a temperature-controlled environment (2261 8C) under a 12 h light:dark schedule with lights off at 15:00 h and given unlimited access to Purina mouse chow and tap water. The animals were acclimated to these conditions for 4 weeks before the experiment. All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co., Ltd. All experiments were performed according to the method described previously [17]. Briefly, hamsters of each group were further subdivided into stress and non-stress groups. The stress protocol was carried out on 5 consecutive days and began at 15:00 h. The stressed hamsters, including the healthy control strain, were subjected daily to 1 h periods of supine immobilization at 4 8C. This consisted of immobilizing the animal by extending their four limbs and taping them to the corners of a small board then putting them in a refrigerator for 1 h. The non-stressed healthy and cardiomyopathic hamsters were not immobilized and just left in their housing cages outside the refrigerator for 1 h. Immediately before and after each stress session as well as the next morning after each session, the hamsters were checked for body weight and to see if they were still alive. Thereafter, we checked the hamsters twice daily until 7 days after the last stress session. Hamsters were autopsied after either being found dead or after decapitation on the final day of the experiment (day 12). In the animals that succumbed to sudden death, 10 h was the maximum time that was allowed to elapse between death and autopsy. Autopsy consisted of removal of the organs and absorption of pleural and peritoneal fluid on a pre-weighed gauze pad. Organs and body cavity fluid (BCF) were then weighed quickly. The surviving animals were sacrificed at the end of the experiment, 7 days after the last stress session, and autopsied as above. Dizocilpine, purchased from Research Biochemicals Incorporated (Natik, MA), was dissolved in physiological
201
saline just before the tests and given intraperitoneally in a volume of 2 ml / kg just prior to the immobilization stress for 5 consecutive days. All results were expressed as mean6S.E.M. Statistical significance of differences in the changes in organ weights was calculated either by using one-way analysis of variance followed by Dunnett’s multiple comparison post-hoc test for multiple dosed groups, or by unpaired Student’s t-test for two group comparison between non-stressed and stressed animals. Mortality results were analyzed by Fisher’s exact probability test. Cumulative surviving percentages of hamsters during the course of the experiments were analyzed using a generalized Wilcoxon test. Neither healthy hamsters with or without stress, nor non-stressed cardiomyopathic hamsters succumbed to sudden death during the course of the experiment (Fig. 1A). In contrast, five out of seven stressed cardiomyopathic hamsters died and there was a statistically significant difference between the mortality of the two groups (P,0.05 by Fisher’s exact probability test). As shown in Fig. 1B, which represents the cumulative surviving percent of hamsters, three animals among the stressed-cardiomyopathic hamsters succumbed during the 5 days of stress sessions, and another two animals died after the stress termination. The difference between the two groups was statistically significant (P,0.05 by generalized Wilcoxon test). Dizocilpine was evaluated for its effect on the sudden death of stressed cardiomyopathic hamsters. Administration of dizocilpine (0.1–10 mg / kg, i.p.) reduced the mortality seen in the stressed-cardiomyopathic hamsters with a bell-shaped dose–response relationship. Dizocilpine provided the maximal protection at 1 mg / kg, with six out of seven animals surviving at the completion of the experiments. However, this change did not reach statistical significance, probably due to the limited number of animals employed for each experimental group (by Fisher’s exact probability test). On the other hand, the highest dose of dizocilpine (10 mg / kg) lost its protective efficacy and most of the animals in that group succumbed during stress sessions, probably because the dose level produced harmful side-effects like hypothermia during stress session which could aggravate the response by cold-immobilization stress. Six days after the completion of the stress sessions, BCF and organ weights were measured (Fig. 2). The stress caused no detectable changes in healthy animals. However, the BCF, heart, adrenal, liver and kidney weights were significantly (P,0.05 by Student’s t-test) increased in the stressed cardiomyopathic hamsters compared with those of non-stressed cardiomyopathic animals. As shown in Fig. 2, administration of dizocilpine significantly prevented increases in the weights of BCF, adrenal, kidney and liver in the stressed-cardiomyopathic hamsters, with statistically significant (P,0.05) differences in the groups dosed with 1 or 10 mg / kg of
202
N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
Fig. 1. (A) Effects of dizocilpine (0.1–10 mg / kg) on sudden death caused by immobilization stress in cardiomyopathic hamsters. Each value represents the percentage of surviving hamsters. Numbers in parentheses indicate the number of animals in each group. C, control non-stressed animals; S, stressed animals. (B) Survival curves of unstressed and stressed cardiomyopathic hamsters receiving dizocilpine treatment. Dizocilpine was administered i.p. just prior to the stress, for 5 consecutive days in the first week. Saline was administered to the vehicle control group.
dizocilpine (Fig. 2). The drug only marginally affected the heart weights. Agreeing with the result of a previous study by this group [17], the present study showed that cold-immobilization stress exerted a lethal effect on cardiomyopathic Syrian hamsters but not on control healthy hamsters, suggesting that the stress had serious and even lethal consequences in cardiovascular dysfunction seen in cardiomyopathic hamsters with a covert heart disease [3,27]. Our previous studies demonstrated that severe arrhythmia
was observed before sudden death in stressed-cardiomyopathic hamsters, implying that acute heart failure and / or lethal arrhythmia might be responsible for the death of the stressed cardiomyopathic hamsters [17]. Supporting this view, heart and adrenal weights were markedly increased in those animals, as confirmed in the present study. It was also supported by results indicating that stress elevated BCF in these animals, an index which is suggestive of the presence of fluid retention in the body cavity commonly seen in animals with congestive heart failure
N. Matsuoka et al. / Brain Research 944 (2002) 200 – 204
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Fig. 2. Effects of dizocilpine on organ weights in stressed cardiomyopathic hamsters. Each column and bar represent the mean6S.E.M. C, control non-stressed animals; S, stressed animals. * P,0.05, ** P,0.01; significantly different compared with saline-treated stressed cardiomyopathic hamsters (by one-way analysis of variance followed by Dunnett’s multiple comparison test). * P,0.05, *** P,0.001; significantly different from non-stressed cardiomyopathic group (by unpaired Student’s t-test). [[ P,0.01, [[[ P,0.001; significantly different from non-stressed healthy animals (by unpaired Student’s t-test).
[20]. Given the fact that the stress produced a marked increase not only in kidney weight but also in serum levels of urea nitrogen and creatinine in the cardiomyopathic hamsters [17], this likely indicates a reduction in glomerular filtration rate of the kidney possibly related to a decrease in myocardial performance, indicative of sudden ventricular dysfunction. The major finding of the present study is that the NMDA receptor antagonist, dizocilpine, prevented sudden death induced by cold-immobilization stress in cardiomyopathic hamsters, and also significantly ameliorated the stressinduced increase in organ weights. These results clearly demonstrate an involvement of NMDA receptors in sudden death of stressed-cardiomyopathic hamsters. We have previously suggested that an activation of the sympathetic nerves triggered by stress participates in the sudden death in this animal model, as a consequence of increased incidence of heart failure and / or cardiac arrhythmia [17]. Though the present study does not elucidate the precise mechanisms of NMDA receptor participation in stressinduced sudden death, it favors the speculation that central NMDA receptors could play a role in the mechanisms by which stress information is conveyed from the brain to the sympathetic nerves [8,24]. Accumulating evidence has suggested that the stimulation of excitatory amino acids receptors in brain areas such as the amygdala, hypothalamus or lower brain areas activate sympathetic nerves and could result in the abnormal changes in cardiovascular function, such as the loss in heart rhythm and even myocardial damage [5,10,12,23].
Pharmacological blockade of glutamate receptors at these sites eliminates a variety of centrally-mediated cardiovascular responses [4,22]. These include baroreceptor reflexes and increases in arterial pressure produced by both stimulation of various brain regions as well as peripheral afferent nerves [22]. There have also been numerous findings suggesting that central glutamatergic neurotransmission plays a pivotal role in stress-induced cardiovascular response. For instance, Soltis and DiMicco showed that hypothalamic injections of NMDA and AMPA receptors antagonists reduced tachycardia induced by stress in rats [26], suggesting an involvement of intrahypothalamic NMDA receptors in that response. Therefore, it is conceivable that activation of NMDA receptors in brain regions like the amygdala, hypothalamus and brain stem could contribute to the etiological cause of sudden death of stressed-cardiomyopathic hamsters as a consequence of sympathetic excitation. The present study cannot rule out the possible involvement of peripheral NMDA receptors located at sympathetic nerves or nerve terminals at vasculature in the heart, kidney and adrenal glands in the preventive effect of dizocilpine on sudden death. In the present study, dizocilpine reduced the increase in adrenal weight produced by stress, indicating that the drug could effectively attenuate sympathetic nerve discharge. This view is supported by earlier findings of other studies, suggesting the involvement of NMDA receptors at the spinal level in the control of sympathetic output [5]. Further investigations will, however, be required to characterize the detailed
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mechanisms by which blockade of NMDA receptors by dizocilpine results in the prevention of sudden death in stressed-cardiomyopathic hamsters. We have previously demonstrated, using the same model, that dopamine D1 / 5 receptors could be involved in the sudden death of cardiomyopathic hamsters [1]. Therefore, it would also be fascinating to delineate the relationship of D1-mediated mechanisms and NMDA-mediated ones in regulating stress-induced cardiovascular malfunction. Dopaminergic and glutamatergic transmissions have long been known to interact at multiple levels in the striatum and in midbrain dopaminergic areas (substantia nigra and ventral tegmental area) [7]. The interaction could be involved in stress-response, since NMDA receptors have been known to play as important signals of stressinduced enhancement in dopaminergic neurotransmission in certain brain areas [6,11]. One of the dopamine-glutamate interactions is represented by the reciprocal modulation of each other’s release at synaptic terminals. However, it has been recently shown that an activation of D1 receptors, but not of D2 receptors, potentiates transsynaptic responses mediated by activation of NMDA receptors [19]. This specific mode of D1-NMDA interaction may partly explain the neural mechanisms involved in the stressinduced cardiac sudden death. In conclusion, the present results have shown that the blockade of NMDA receptors by dizocilpine significantly prevented sudden death of cardiomyopathic hamsters induced by the cold-immobilization stress, suggesting that NMDA receptors have an important role in the etiology of stress-induced cardiac sudden death of cardiomyopathic hamsters. These findings provide the first experimental evidence for the potential therapeutic value of NMDA antagonists for cardiac sudden death associated with stress.
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