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Changes in extracellular calcium concentration in the immature rat cerebral cortex during anoxia are not influenced by MK-801
DEVELOPMENTAL BRAIN RESEARCH ELSEVIER
Developmental Brain Research 77 (1994) 146-150
,
Short Communication
Changes in extracellular calcium concentration in the immature rat cerebral cortex during anoxia are not influenced by MK-801 Malgorzata Puka-Sundvall a, Henrik Hagberg a,b, Peter Andin6 a,, a Department of Anatomy and Cell Biology, Institute ofNeurobiology, Unicersityof G6teborg, Medicmaregatan 3-5, S 413 90, G6teborg, Sweden b Department of Obstetrics and Gynecology, Unicersity of G&eborg, G&eborg, Sweden (Accepted 19 October 1993)
Abstract
The extracellular calcium concentration (tea 2+ ]ec) was recorded by calcium-sensitive microelectrodes in the parietal cortex of 9-11 day old rats during anoxia. During the first 10 min of anoxia, [Ca2+]ec increased from 1.1 mM to 1.5 + 0.23 raM, and thereafter it started to decrease reaching below basal level after around 13 min. The [Ca2+]e~ decrease was either slow and continuous, or biphased with a rapid initial decrease followed by a continuous slow decrease. After 60 min of anoxia, the tea 2+]ec had reached 0.2-0.3 mM. Changes in [Ca2+]ec in animals treated with the NMDA receptor antagonist MK-801 (0:3 mg/kg i.p.) did not display any significant differences compared to controls. Thus, the strong neuroprotective effect of MK-801 in ischemic situations in the immature brain can not be explained by a prevention of calcium entry during anoxic depolarization. Key words: Anoxia; Hypoxia; Excitatory amino acid; NMDA receptor; Calcium: MK-801
There is substantial evidence for that excitatory amino acid- (EAA) mediated intracellular calcium load is involved in the development of ischemic damage in the adult brain [7,8,29]. Less is known about the immature brain, however, data indicate the same important role for EAA and calcium [14,15,21,22,27,32], e.g., in a model of hypoxic-ischemia in 7 day old rats, EAAs and glycine are released to the extracellular space [3], calcium is accumulated in brain regions that will eventually undergo infarction [37], and neurons that express EAA receptors seem to be particularly vulnerable [35]. Furthermore, a number of compounds that counteract calcium entry offers neuroprotection in the neonatal rat model: the calcium-entry blocker flunarizine [34], kynurenic acid [2] and kynurenine (by conversion to kynurenic acid) [30] which interact with E A A receptors of both the N M D A (N-methyl-o-aspartate) and nonN M D A types. Finally, a number of studies have documented the strong neuroprotective efficacy of the
* Corresponding author. Fax: (46) 31-88-3330. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 3 ) E 0 1 8 3 - L
N M D A receptor antagonist MK-801 [40] in neonatal rat models [9,16,20,25,31,36,40]. After 2-3 min of anoxia in the adult brain, there is a rapid increase in the extraceUular potassium concentration to 60 mM, a rapid decrease in the extracellular calcium concentration ([CaZ+]ec) to around 0.1 mM [18], and a negative shift in the extracellular reference potential (REF), i.e. the 'anoxic depolarization' (AD). It is well known that the immature brain responds quite differently to anoxia with regard to shifts in extracellular potassium. The onset of AD is delayed and the shift is protracted in the neonatal compared to the adult cerebral cortex [17,24]. The [Ca2+]ec has, to our knowledge, not been studied during AD in the immature brain. Therefore, the present study was performed in order to describe the changes in [Ca2+]ec during anoxia in the immature rat brain, and to investigate whether these changes could be influenced by MK-801, thereby giving a clue to the mechanisms of N M D A receptor-mediated toxicity during anoxia. Calcium-sensitive microelectrodes for recordings of [Ca2 +]ec and R E F was constructed as described previously [1,4]. Sprague-Dawley rats at the age of 9-11 days were anesthetized with halothane (2% for induction and 0.8-1.0 for maintainance) in a oxygen/nitrous
M. Puka-Sundvall et al. / Det~elopmental Brain Research 77 (1994) 146-150
oxide mixture (1 : 1). The rectal temperature was monitored by a rectal probe (Fluke, 51 K / J ) and kept at 37°C by a heating lamp and pad. The skull over the right parietal cortex was exposed and a hole with a diameter of 2 - 3 mm was drilled in the bone. The dura was incised and the cortical surface was covered with a modified Ringer solution (1.3 mM CaCI2, 3 mM KC1, 145 mM NaCI). The animal was put in a stereotaxic apparatus made from paris mold. The calcium-electrode was calibrated in vitro (electrode responses varied between 23 and 27 mV change per decade shift in external calcium concentration) and then in the 1.3 mM CaZ+-containing solution on the cortical surface. Thereafter, the calcium-electrode was lowered 0.7 mm down into the parietal cortex (anterior-posterior: exactly between lambda and bregma; lateral: 2 mm). After obtaining stable recordings of [Ca 2+ ]ec and REF, anoxia was induced by shifting the inhalation gas to 100% nitrogen. Recordings were continued for 60 min after the onset of anoxia. Two hours before the onset of anesthesia, i.e. approximately 3 h before anoxia, five rats were treated with 0.3 m g / k g MK-801 intraperitoneally ('MK-801 group'). They were compared to six controls, that did not recieve MK-801 ('control group'), with regard to anoxic shifts in [Ca2+]ec and REF. The non-parametric unpaired two-tailed Mann-Whitney U-test and the two-tailed repeated measures A N O V A test were used for statistical calculations. Animals that recieved 0.3 m g / k g MK-801 did not differ from controls with regard to rectal temperature or respiratory pattern neither before nor during surgery. The age and weight of the rats were 10.2 ___0.4 days and 17.5 _+ 1.1 g, and 10.6 _+ 0.2 days and 16.7 + 1.0 g in the control and MK-801 group, respectively. The basal [Ca2+]ec in the cortex was 1.11 + 0.09 mM in the control group and 1.18+_0.11 mM in the MK-801 group. As the basal levels displayed very little variation, the basal level before anoxia was standardized to 1.1 mM in all animals and the anoxic shifts were compared to this level. Upon nitrogen inhalation, spontaneous respiration ceased within 60 s in both the control and MK-801 group. During the first 10 rain period of anoxia, the [Ca2+]~ showed a transient increase to 1.53 + 0.23 mM in the control group and to 1.79 +_ 0.16 mM in the MK-801 group (no statistically significant difference) (Fig. 1). Thereafter, the [Ca2+]e~ decreased over the following 50 min of recording, finally reaching 0.29 + 0.05 and 0.23 + 0.05 mM in the control and MK-801 group, respectively. This decrease of [Ca 2+]~c displayed two different patterns. First, in 3 / 6 control and 3 / 5 MK-801 animals, the decrease was slow and gradual over 50 min (Fig. 2A). Second, in 3 / 6 control and 2 / 5 MK-801 animals, there was a period of rapid decrease after 14 + 2.1 rain of anoxia, followed by a continuous slow decrease (Fig. 2B). The R E F displayed a slow negative shift over the recording
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Duration of anoxia (min) Fig. 1. The extracellular calcium concentration ([Ca 2+ ]ec) after different durations of anoxia. The calcium-electrode was positioned in the parietal cortex and anoxia was induced by nitrogen inhalation of the rat. After a transient increase in [Ca 2+ ]~,,, it slowly decreased to an approximate steady-state concentration of 0.2-0.3 raM. There were no significant differences in the control group (n = 6) and the group of animals that had recieved 0.3 m g / k g of MK-801 3 h prior to anoxia (n = 5). Data are presented as mean_+ S.E.M.
period of 9.8 _+ 1.7 and 85 + 0.8 mV in the control and MK-801 group, respectively. The rectal temperature was 37.1 _+ 0.15°C and 37.0 + 0.09°C at the start of anoxia, and 36.5 _+ 0.29°C and 37.1 + 0.22°C after 60 min of anoxia in the control and MK-801 group, respectively. There were no significant differences between the control and MK-801 groups with regard to [Ca2+]ec, R E F or temperature shifts. The present study showed that during anoxia in the immature brain, the AD was accompanied by a decrease in [CaZ+]ec . In accordance with a delayed AD in immature brain, the decrease in [Ca2+]ec occurred after longer duration of anoxia and was slower than what is found in the mature brain. The N M D A receptor antagonist MK-801 did not influence the anoxic decrease in [Ca2+]ec Hansen [17] and Mares and coworkers [24] described the shift in extracellular potassium during cerebral anoxia in rats of different ages. The increase in extracellular potassium could be divided into different phases, and the duration of these phases were longer in the very immature rats. First, a slow increase was seen followed by a sudden rapid increase to about 70 raM. Then it slowly increased until reaching a steady-state of about 90 raM. In the two studies, rats of the same age as used in the present study (9-11 days), displayed the phase of rapid increase after 8-10 min [17] and 12-15 min [24]. This is in agreement with our finding of 14 rain of anoxia until the rapid phase of [Ca2+]ec decrease occurred. However, we did only see a rapid phase in 5 out of 11
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M. Puka-Sundt,all et al. / Del'elopmental Brain Research 77 ~I994) 146-1.50
animals, and such a variability was not described for potassium [17,24]. During the initial phase of anoxia, when extracellular potassium is slowly increasing, we observed a transient increase in [Ca2+]ec . This increase was enhanced in animals treated with MK-801 (although not statistically significant). One obvious reason for the increase is a shrinkage of the extracellular space due to intracellular edema [18]. In the adult brain, the rapid decrease in [Ca2+]ec is in fact also preceeded by an increase [18]. However, this increase is only of about 0.1 mM compared to 0.4 and 0.7 m M in the control and MK-801 group, respectively, in our immature rats. The immature cerebral cortex has a larger extracellular space [5] and may thus undergo more severe degree of shrinkage, seen as a more pronounced increase in [Ca2+]ec. Another possibility would be an increased extrusion of calcium ions from the cells via the sodium-calcium exchanger, as the energy reserves are preserved for
longer time periods in the immature than in the adult brain [12]. Indeed the intracellular calcium concentration has been shown to increase before the onset of anoxic depolarization [39] which may cause increased extrusion. The effect of MK-801 can only be speculated upon. Although the extracellular levels of EAAs arc not elevated until the onset of AD [33], normal levels of EAAs may cause a minor inflow of calcium ions through N M D A receptor activated channels already during the initial anoxic phase, that is not fully counteracted by the sodium-calcium exchanger. This relatively small influx of calcium ions would not be reflected as a decrease in [Ca2+]~c due to shrinkage of the extracellular space. A block of this initial calcium influx by MK-801 could thus be seen as an enhancement of the shrinkage-induced increase in [Ca ~'+]~c. Doses of more than 0.3 m g / k g of MK-801 could not be given to the immature rats, as it caused respiratory arrest during anesthesia. However, this dose should be
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10 min Fig. 2. Typical recordings of the extracellular calcium concentration ([Ca 2 + ]ec) and reference potential (REF) in the parietal cortex of l0 day old rats during 60 min of anoxia. Two typical patterns of shifts were seen. A: a transient increase in [Ca2+]+c, followed by a slowly ongoing decrease reaching a steady-state concentration after 50-60 min. B: a transient increase in [CaZ+]cc followed by a rapid phase of decrease in [Ca2+]cc, and finally a continuous slow decrease reaching a steady-state after 50-60 min. In both cases, the R E F displayed a negative shift in parallell with the decrease in [cae+]ec . Arrow indicate onset of anoxia.
M. Puka-SundL~all et al. / De~elopmen tal Brain Research 77 (1994) 146-150
sufficient to block NMDA receptors, as it reduces NMDA toxicity in neonates [28]. In addition, 0.3 m g / k g of MK-801 is neuroprotective after cerebral ischemia in adults [10], and reduces hemispheric infarction by 60% after neonatal hypoxic-ischemia [16]. The decrease of [Ca2+]~¢ during anoxia is generally believed to reflect a cellular uptake of calcium [19]. In the adult brain, MK-801 does not influence the time until AD or the changes in extracellular potassium [23]; however, it seems to delay the decrease in [Ca2+]e~ [41] after cardiac arrest, and to prevent the rise of intracellular calcium concentration in a focal ischemic lesion [13]. A lot of data implicate that the immature brain is hypersensitive to stimulus of NMDA receptors [27]. The density of NMDA receptors is transiently high in the immature brain [37], the NMDA receptor is more easily activated in immature brain due to less magnesium block [6], intracerebral injections of NMDA cause larger damage in the immature brain than in the adult [26], and finally NMDA receptor antagonists are highly neuroprotective in neonatal hypoxic-ischemia [9,20,25, 31,36] exceeding the effect of non-NMDA receptor antagonists [16]. Therefore, one might have suspected an ability of MK-801 to inhibit the cellular uptake of calcium during anoxia in the immature brain. Thus, there must be other mechanisms, besides blocking calcium entry during AD, behind the neuroprotective effect of MK-801 in immatures. As has been indicated in adult brain, postischemic and penumbral events related to calcium entry may be important [4,10,29], and MK-801 is also neuroprotective when administered after the hypoxic-ischemic insult in neonates [16,20]. This should be focused upon in future studies. This work was supported by the Swedish Medical Research Council (9455), the Medical Faculty of the University of G6teborg, the Sven Jerring Foundation, the 1987 Foundation for Stroke Research, the Ake Wiberg Foundation, the Ahlen Foundation, the Magnus Bergwall Foundation, the Laerdal Foundation, the Konung Gustav V's 80 ~r Foundation, the First May Flower Annual Campaign. M. PukaSundvall was supported by the Swedish Institute.
[1] Andin~, P., Jacobson, I. and Hagberg, H., Calcium uptake evoked by electrical stimulation is enhanced postischemically and precedes delayed neuronal death in CA1 of rat hippocampus: involvement of N-methyI-D-aspartate receptors, J. Cereb. Blood Flow Metab., 8 (1988) 799-807. [2] Andin~, P., Lehmann, A., Ellr~n, K., Wennberg E., Kjellmer, I., Nielsen, T. and Hagberg, H., The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotection, Neurosci. Lett., 90 (1988b) 2(18- 212. [3] Andin6, P., Sandberg, M., Bhgenholm, R., Lehmann, A. and Hagberg, H., Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia, Dec. Brain Res., 64 (1991) 115-120. [4] Andin6, P., Jacobson, 1. and Hagberg, H., Enhanced calcium uptake by CAI pyramidal cell dendrites in the postischemic
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phase despite subnormal evoked field potentials: excitatory amino acid receptor dependency and relationship to neuronal damage, J. Cereb. Blood Flow Metab., 12 (1992) 773-783. [5] Bondareff, W. and Pysh, J.J., Distribution of the extracellular space during postnatal maturation of rat cerebral cortex, Anat. Rec., 160 (1969) 773-780. [6] Bowe, M.A. and Nadler, J.V., Developmental increase in the sensitivity to magnesium of NMDA receptors on CAI hippocampal pyramidal cells, Del:. Bran Res., 56 (19901 55-61. [7] Choi, D.W., Methods for antagonizing glutamate neurotoxicity, Cerebrol,asc. Brain Metab. Bet'., 2 (1990) 105-147. [8] Diemer, N.-H., Johansen, F.F. and Jorgensen, M.B., N-methylD-aspartate and non-N-methyl-D-aspartate antagonists in global cerebral ischemia, Stroke, 21 (1990) III-39-111-42. [9] Ford, L.M., Sanberg, P.R., Norman, A.B. and Fogelson, M.H., MK-801 prevents hippocampal neurodegeneration in neonatal hypoxic-ischemic rats, Arch. Neurol., 46 (1989) 1090-1096. [10] Gill, R., Foster, A.C. and Woodruff, G.N., MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period, Neuroscience, 25 (1988) 847-855. [11] Gill, R., Andin~ P., Hillered, L., Persson, L. and Hagberg, H., The effect of MK-801 on cortical spreading depression in the penumbral zone following focal ischaemia in the rat, J. Cereb. Blood Flow Metab., 12 (1992) 371-379. [12] Gonzfilez-Mendez, R., McNeill, A., Gregory, G.A., Wall, S.D., Gooding, C.A., Litt, L. and James, T.L., Effects of hypoxic hypoxia on cerebral phosphate metabolites and pH in the anesthetized infant rabbit, J. Cereb. Blood Flow Metab., 5 (19851 512-516. [13] Greenberg, J.H., Uematsu, D., Araki, N., Hickey, W.F. and Reivich, M., Cytosolic free calcium during focal cerebral ischemia and the effects of nimodipine on calcium and histologic damage, Stroke, 21, Suppl. IV (1990) IV-72 IV-77. [14] Hagberg, H., Andin~ P. and Lehmann, A., Excitatory, amino acids and hypoxic-ischemic damage in the immature brain. In A. Schurr and B.M. Rigor (Eds.), Cerebral lschemia and Resuscitation, CRC Press, Boca Raton, 1900, pp. 165-177. [15] Hagberg, H., Hypoxic-ischemic damage in the neonatal brain: excitatory amino acids, Der. Pharmacol. Ther., 18 (1993) 139144. [16] Hagberg, H., Diemer, N.-H., Gilland, E. and Andin& P., Hypoxic-ischemic damage in the immature brain: effect of NMDAand non-NMDA receptor antagonists, submitted. [17] Hansen, A.J., Extracellular potassium concentration in juvenile and adult rat brain cortec during anoxia, Aeta Physiol. Scand., 99 (1976) 412-420. [18] Hansen, A.J., Effect of anoxia on ion distribution in the brain, Physiol. Re~,., 65 (1985) 101-148. [19] Harris, R.J., Symon, L., Branston, N.M. and Bayhan, Changes in extracellular calcium activity in cerebral ischemia, Z Cereb. Blood Flow Metab., 1 (1981) 203-209. [20] Hattori, H., Morin, A.M., Schwartz, P.H., Fujikawa, D.G. and Wasterlain, C.G., Posthypoxic treatment with MK-801 reduces hypoxic-ischemic damage in the neonatal rat, Neurology, 39 (1989) 713-718. [21] Hattori, H. and Wasterlain, C.G., Excitatory amino acids in the developing brain: ontogeny, plasticity, and excitotoxicity, Pediatr. Neurol., 6 (1990) 219-228. [22] Johnston, M.V., Silverstein, F.S., Perinatal anoxia. In J.T. Coyle (Ed.), Animal Models of Dementia, Allan R. Liss, New York, 1987, pp. 223-251. [23] Lauriten, M. and Hansen, A.J., The effect of glutamate receptor blockade on anoxic depolarization and cortical spreading depression, J. Cereb. Blood Flow Metab., 12 (1992) 223-229. [24] Mares, P., Kriz, N., Brozek, G. and Bures, J., Anoxic changes of extracellular potassium concentration in the cerebral cortex of young rats, Exp. Neurol., 53 (1976) 12 20.
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[25] McDonald, J.W., Silverstein, F.S. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359-361. [26] McDonald, J.W., Silverstein, F.S. and Johnston, M.V., Neurotoxicity of N-methyI-D-aspartate is markedly enhanced in developing rat central nervous system, Brain Res., 459 (1988) 200-203. [27] McDonald, J.W. and Johnston, M.V., Physiological and pathophysiological roles of excitatory amino acids during central nervous system development, Brain Res. Rec., 15 (1990) 41-70. [28] McDonald, J.W., Silverstein, F.S., Cardona, D., Hudson, C., Chien, R. and Johnston, M.V., Systemic administration of MK801 protects against N-methyl-D-aspartate- and quisqualatemediated neurotoxicity in perinatal rats, Neuroscience, 36 (1990) 589-599. [29] Meldrum, B., Protection against ischaemic neuronal damage by drugs actin on excitatory neurotransmission, Cerebrot,ase. Brain Metab. Ree., 2 (1990) 27-57. [30] Nozaki, K. and Beal, M.F., Neuroprotective effects of l,kynurenine on hypoxia-ischemia and NMDA lesions in neonatal rats, .L Cereb. Blood Flow Metab., 12 (1992) 400-407. [31] Olney, J.W., lkonomidou, J.L., Mosinger, J.L. and Frierdich, G,, MK-801 prevents hypobaric-ischemic neuronal degeneration in infant rat brain, J, Neurosci., 9 (1989) 1701-1704. [32] Olney, J.W., Excitotoxicity and ischemic brain damage in infancy and adulthood. In J. Krieglstein and H. Oberpichler (Eds.), Pharmacology of Cerebral Ischemia, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 1990, pp. 153-168. [33] P~rez-Pin6n, M.A., Nilsson, G.E. and Lutz P.L., Relationship between ion gradients and neurotransmitter release in the newborn rat striatum during anoxia, Brain Res., 602 (1993) 228-233.
[34] Silverstein, F.S., Buchanen, K., itudson. C. and ,Iolanslon, M.V., Flunarizine limits hypoxia-ischemia induced morphologic injury, in immature rat brain, Stroke, 17 (1986) 477-482 [35] Silverstein, F.S., Torke, L, Barks, J. and Johnston, M.V., tty-. poxia - ischemia produces focal disruption of glutamate' receptors in developing brain, Dec. Brain Res.. 34 (IC)87) 3 3 39. [36] Silverstein, F.S., McDonald, J.W., Bommarito, M. and Johnstoi~, M.V., Effects of hypoxia-isehemia and MK-801 treatment on the binding of a phencyclidine analogue in the developing rat brain. Stroke, 21 (1990) 311t-315. [37] Stein, D.T. and Vannucci, R.C., Calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat, J. Cereb. Blood Flow Metab., 8 (1988) 834-842. [38] Tremblay, E., Roisin, M.P., Represa, A., Charriaut-Marlangue, C. and Ben-Ari, Y., Transient increased NMDA binding sites in the developing rat hippocampus, Brain Res., 461 (1988)393-396. [39] Uematsu, D., Greenberg, J.H., Reivich, M., Kobayashi, S. and Karp, A., In vivo fluorometric measurment of changes in cytosolic free calcium from the cat cortex during anoxia. ,1. Cereb. Blood Flow Metab., 8 (1988) 367-374. [40] Wong, E.H.F., Kemp, J.A., Priestly, T., Knight, A.R., Woodruff, G.N. and lversen, EL., The anticonvulsant MK-801 is a potent N-methyI-D-aspartate antagonist, Proc. Natl. Acad. Sci. USA, 83 (1986) 7104-71(18. [41] Xie, Y., Zacharias, E., Dengler, K., Wilffert, B. and Tegtmeier, F., Blockade of glutamate receptors but not voltage-gated Ca 2~ channels attenuates ischemia-induced decline of extracellular calcium. In J. Kriglstein and H. Oberpichler-Schwenk (Eds.), 4th International Symposium on Pharmacology of Cerebral lschemia (abstract book), 1992, p. 46 (DE2).
Developmental Brain Research 77 (1994) 146-150
,
Short Communication
Changes in extracellular calcium concentration in the immature rat cerebral cortex during anoxia are not influenced by MK-801 Malgorzata Puka-Sundvall a, Henrik Hagberg a,b, Peter Andin6 a,, a Department of Anatomy and Cell Biology, Institute ofNeurobiology, Unicersityof G6teborg, Medicmaregatan 3-5, S 413 90, G6teborg, Sweden b Department of Obstetrics and Gynecology, Unicersity of G&eborg, G&eborg, Sweden (Accepted 19 October 1993)
Abstract
The extracellular calcium concentration (tea 2+ ]ec) was recorded by calcium-sensitive microelectrodes in the parietal cortex of 9-11 day old rats during anoxia. During the first 10 min of anoxia, [Ca2+]ec increased from 1.1 mM to 1.5 + 0.23 raM, and thereafter it started to decrease reaching below basal level after around 13 min. The [Ca2+]e~ decrease was either slow and continuous, or biphased with a rapid initial decrease followed by a continuous slow decrease. After 60 min of anoxia, the tea 2+]ec had reached 0.2-0.3 mM. Changes in [Ca2+]ec in animals treated with the NMDA receptor antagonist MK-801 (0:3 mg/kg i.p.) did not display any significant differences compared to controls. Thus, the strong neuroprotective effect of MK-801 in ischemic situations in the immature brain can not be explained by a prevention of calcium entry during anoxic depolarization. Key words: Anoxia; Hypoxia; Excitatory amino acid; NMDA receptor; Calcium: MK-801
There is substantial evidence for that excitatory amino acid- (EAA) mediated intracellular calcium load is involved in the development of ischemic damage in the adult brain [7,8,29]. Less is known about the immature brain, however, data indicate the same important role for EAA and calcium [14,15,21,22,27,32], e.g., in a model of hypoxic-ischemia in 7 day old rats, EAAs and glycine are released to the extracellular space [3], calcium is accumulated in brain regions that will eventually undergo infarction [37], and neurons that express EAA receptors seem to be particularly vulnerable [35]. Furthermore, a number of compounds that counteract calcium entry offers neuroprotection in the neonatal rat model: the calcium-entry blocker flunarizine [34], kynurenic acid [2] and kynurenine (by conversion to kynurenic acid) [30] which interact with E A A receptors of both the N M D A (N-methyl-o-aspartate) and nonN M D A types. Finally, a number of studies have documented the strong neuroprotective efficacy of the
* Corresponding author. Fax: (46) 31-88-3330. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 3 8 0 6 ( 9 3 ) E 0 1 8 3 - L
N M D A receptor antagonist MK-801 [40] in neonatal rat models [9,16,20,25,31,36,40]. After 2-3 min of anoxia in the adult brain, there is a rapid increase in the extraceUular potassium concentration to 60 mM, a rapid decrease in the extracellular calcium concentration ([CaZ+]ec) to around 0.1 mM [18], and a negative shift in the extracellular reference potential (REF), i.e. the 'anoxic depolarization' (AD). It is well known that the immature brain responds quite differently to anoxia with regard to shifts in extracellular potassium. The onset of AD is delayed and the shift is protracted in the neonatal compared to the adult cerebral cortex [17,24]. The [Ca2+]ec has, to our knowledge, not been studied during AD in the immature brain. Therefore, the present study was performed in order to describe the changes in [Ca2+]ec during anoxia in the immature rat brain, and to investigate whether these changes could be influenced by MK-801, thereby giving a clue to the mechanisms of N M D A receptor-mediated toxicity during anoxia. Calcium-sensitive microelectrodes for recordings of [Ca2 +]ec and R E F was constructed as described previously [1,4]. Sprague-Dawley rats at the age of 9-11 days were anesthetized with halothane (2% for induction and 0.8-1.0 for maintainance) in a oxygen/nitrous
M. Puka-Sundvall et al. / Det~elopmental Brain Research 77 (1994) 146-150
oxide mixture (1 : 1). The rectal temperature was monitored by a rectal probe (Fluke, 51 K / J ) and kept at 37°C by a heating lamp and pad. The skull over the right parietal cortex was exposed and a hole with a diameter of 2 - 3 mm was drilled in the bone. The dura was incised and the cortical surface was covered with a modified Ringer solution (1.3 mM CaCI2, 3 mM KC1, 145 mM NaCI). The animal was put in a stereotaxic apparatus made from paris mold. The calcium-electrode was calibrated in vitro (electrode responses varied between 23 and 27 mV change per decade shift in external calcium concentration) and then in the 1.3 mM CaZ+-containing solution on the cortical surface. Thereafter, the calcium-electrode was lowered 0.7 mm down into the parietal cortex (anterior-posterior: exactly between lambda and bregma; lateral: 2 mm). After obtaining stable recordings of [Ca 2+ ]ec and REF, anoxia was induced by shifting the inhalation gas to 100% nitrogen. Recordings were continued for 60 min after the onset of anoxia. Two hours before the onset of anesthesia, i.e. approximately 3 h before anoxia, five rats were treated with 0.3 m g / k g MK-801 intraperitoneally ('MK-801 group'). They were compared to six controls, that did not recieve MK-801 ('control group'), with regard to anoxic shifts in [Ca2+]ec and REF. The non-parametric unpaired two-tailed Mann-Whitney U-test and the two-tailed repeated measures A N O V A test were used for statistical calculations. Animals that recieved 0.3 m g / k g MK-801 did not differ from controls with regard to rectal temperature or respiratory pattern neither before nor during surgery. The age and weight of the rats were 10.2 ___0.4 days and 17.5 _+ 1.1 g, and 10.6 _+ 0.2 days and 16.7 + 1.0 g in the control and MK-801 group, respectively. The basal [Ca2+]ec in the cortex was 1.11 + 0.09 mM in the control group and 1.18+_0.11 mM in the MK-801 group. As the basal levels displayed very little variation, the basal level before anoxia was standardized to 1.1 mM in all animals and the anoxic shifts were compared to this level. Upon nitrogen inhalation, spontaneous respiration ceased within 60 s in both the control and MK-801 group. During the first 10 rain period of anoxia, the [Ca2+]~ showed a transient increase to 1.53 + 0.23 mM in the control group and to 1.79 +_ 0.16 mM in the MK-801 group (no statistically significant difference) (Fig. 1). Thereafter, the [Ca2+]e~ decreased over the following 50 min of recording, finally reaching 0.29 + 0.05 and 0.23 + 0.05 mM in the control and MK-801 group, respectively. This decrease of [Ca 2+]~c displayed two different patterns. First, in 3 / 6 control and 3 / 5 MK-801 animals, the decrease was slow and gradual over 50 min (Fig. 2A). Second, in 3 / 6 control and 2 / 5 MK-801 animals, there was a period of rapid decrease after 14 + 2.1 rain of anoxia, followed by a continuous slow decrease (Fig. 2B). The R E F displayed a slow negative shift over the recording
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Duration of anoxia (min) Fig. 1. The extracellular calcium concentration ([Ca 2+ ]ec) after different durations of anoxia. The calcium-electrode was positioned in the parietal cortex and anoxia was induced by nitrogen inhalation of the rat. After a transient increase in [Ca 2+ ]~,,, it slowly decreased to an approximate steady-state concentration of 0.2-0.3 raM. There were no significant differences in the control group (n = 6) and the group of animals that had recieved 0.3 m g / k g of MK-801 3 h prior to anoxia (n = 5). Data are presented as mean_+ S.E.M.
period of 9.8 _+ 1.7 and 85 + 0.8 mV in the control and MK-801 group, respectively. The rectal temperature was 37.1 _+ 0.15°C and 37.0 + 0.09°C at the start of anoxia, and 36.5 _+ 0.29°C and 37.1 + 0.22°C after 60 min of anoxia in the control and MK-801 group, respectively. There were no significant differences between the control and MK-801 groups with regard to [Ca2+]ec, R E F or temperature shifts. The present study showed that during anoxia in the immature brain, the AD was accompanied by a decrease in [CaZ+]ec . In accordance with a delayed AD in immature brain, the decrease in [Ca2+]ec occurred after longer duration of anoxia and was slower than what is found in the mature brain. The N M D A receptor antagonist MK-801 did not influence the anoxic decrease in [Ca2+]ec Hansen [17] and Mares and coworkers [24] described the shift in extracellular potassium during cerebral anoxia in rats of different ages. The increase in extracellular potassium could be divided into different phases, and the duration of these phases were longer in the very immature rats. First, a slow increase was seen followed by a sudden rapid increase to about 70 raM. Then it slowly increased until reaching a steady-state of about 90 raM. In the two studies, rats of the same age as used in the present study (9-11 days), displayed the phase of rapid increase after 8-10 min [17] and 12-15 min [24]. This is in agreement with our finding of 14 rain of anoxia until the rapid phase of [Ca2+]ec decrease occurred. However, we did only see a rapid phase in 5 out of 11
148
M. Puka-Sundt,all et al. / Del'elopmental Brain Research 77 ~I994) 146-1.50
animals, and such a variability was not described for potassium [17,24]. During the initial phase of anoxia, when extracellular potassium is slowly increasing, we observed a transient increase in [Ca2+]ec . This increase was enhanced in animals treated with MK-801 (although not statistically significant). One obvious reason for the increase is a shrinkage of the extracellular space due to intracellular edema [18]. In the adult brain, the rapid decrease in [Ca2+]ec is in fact also preceeded by an increase [18]. However, this increase is only of about 0.1 mM compared to 0.4 and 0.7 m M in the control and MK-801 group, respectively, in our immature rats. The immature cerebral cortex has a larger extracellular space [5] and may thus undergo more severe degree of shrinkage, seen as a more pronounced increase in [Ca2+]ec. Another possibility would be an increased extrusion of calcium ions from the cells via the sodium-calcium exchanger, as the energy reserves are preserved for
longer time periods in the immature than in the adult brain [12]. Indeed the intracellular calcium concentration has been shown to increase before the onset of anoxic depolarization [39] which may cause increased extrusion. The effect of MK-801 can only be speculated upon. Although the extracellular levels of EAAs arc not elevated until the onset of AD [33], normal levels of EAAs may cause a minor inflow of calcium ions through N M D A receptor activated channels already during the initial anoxic phase, that is not fully counteracted by the sodium-calcium exchanger. This relatively small influx of calcium ions would not be reflected as a decrease in [Ca2+]~c due to shrinkage of the extracellular space. A block of this initial calcium influx by MK-801 could thus be seen as an enhancement of the shrinkage-induced increase in [Ca ~'+]~c. Doses of more than 0.3 m g / k g of MK-801 could not be given to the immature rats, as it caused respiratory arrest during anesthesia. However, this dose should be
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10 min Fig. 2. Typical recordings of the extracellular calcium concentration ([Ca 2 + ]ec) and reference potential (REF) in the parietal cortex of l0 day old rats during 60 min of anoxia. Two typical patterns of shifts were seen. A: a transient increase in [Ca2+]+c, followed by a slowly ongoing decrease reaching a steady-state concentration after 50-60 min. B: a transient increase in [CaZ+]cc followed by a rapid phase of decrease in [Ca2+]cc, and finally a continuous slow decrease reaching a steady-state after 50-60 min. In both cases, the R E F displayed a negative shift in parallell with the decrease in [cae+]ec . Arrow indicate onset of anoxia.
M. Puka-SundL~all et al. / De~elopmen tal Brain Research 77 (1994) 146-150
sufficient to block NMDA receptors, as it reduces NMDA toxicity in neonates [28]. In addition, 0.3 m g / k g of MK-801 is neuroprotective after cerebral ischemia in adults [10], and reduces hemispheric infarction by 60% after neonatal hypoxic-ischemia [16]. The decrease of [Ca2+]~¢ during anoxia is generally believed to reflect a cellular uptake of calcium [19]. In the adult brain, MK-801 does not influence the time until AD or the changes in extracellular potassium [23]; however, it seems to delay the decrease in [Ca2+]e~ [41] after cardiac arrest, and to prevent the rise of intracellular calcium concentration in a focal ischemic lesion [13]. A lot of data implicate that the immature brain is hypersensitive to stimulus of NMDA receptors [27]. The density of NMDA receptors is transiently high in the immature brain [37], the NMDA receptor is more easily activated in immature brain due to less magnesium block [6], intracerebral injections of NMDA cause larger damage in the immature brain than in the adult [26], and finally NMDA receptor antagonists are highly neuroprotective in neonatal hypoxic-ischemia [9,20,25, 31,36] exceeding the effect of non-NMDA receptor antagonists [16]. Therefore, one might have suspected an ability of MK-801 to inhibit the cellular uptake of calcium during anoxia in the immature brain. Thus, there must be other mechanisms, besides blocking calcium entry during AD, behind the neuroprotective effect of MK-801 in immatures. As has been indicated in adult brain, postischemic and penumbral events related to calcium entry may be important [4,10,29], and MK-801 is also neuroprotective when administered after the hypoxic-ischemic insult in neonates [16,20]. This should be focused upon in future studies. This work was supported by the Swedish Medical Research Council (9455), the Medical Faculty of the University of G6teborg, the Sven Jerring Foundation, the 1987 Foundation for Stroke Research, the Ake Wiberg Foundation, the Ahlen Foundation, the Magnus Bergwall Foundation, the Laerdal Foundation, the Konung Gustav V's 80 ~r Foundation, the First May Flower Annual Campaign. M. PukaSundvall was supported by the Swedish Institute.
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