Brain Research, 528 (1990) 133-137 Elsevier

133

BRES 24281

Ischemic brain injury in vitro: protective effects of NMDA receptor antagonists and calmidazolium Roman Pohorecki, Gerald L. Becker, Pamela J. Reilly and Dennis F. Landers Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE (U.S.A.) (Accepted 5 June 1990) Key words: Ischemia; Anoxia; Adenosine triphosphate; MK801; Ketamine; Calmidazolium; Calcium

Excessive Ca2+ influx through NMDA receptor-coupled channels has been linked to neuronal cell death. Using an in vitro model of transient brain ischemia, we investigated possible protective effects of NMDA receptor antagonists ketamine or MK-801 and of calmidazolium, an inhibitor of intracellular Ca2+-activated proteins. Brain ischemia/recovery was simulated in isolated hippocampal slices and injury monitored by measurement of ATP levels. Omission of both glucose and oxygen (but not oxygen alone) for 20 min led to persistent ATP deficits after 4 h recovery, Addition of ketamine or MK-801 at 1/~M permitted ATP to recover within 1 h, as did addition of calmidazolium at 10/~M. Our findings are consistent with other reports that NMDA receptor antagonists can protect neuronal tissue from ischemic damage. The role of inappropriately activated Ca2÷-mediated signaling processes in the mechanism(s) of such injury is suggested by the protection also seen with calmidazolium, an inhibitor of calmodulin and other structurally related proteins such as calpain(s) and protein kinase C. The inhibition of intracellular Ca2+ target proteins may be an alternative for protection of the brain against injury due to insults that activate NMDA receptors. Glutamate and certain other amino acids are abundant excitatory neurotransmitters in the central nervous system 1. On the basis of binding affinity, 4 classes of glutamate/EAA receptors may be distinguished: kainate, quisqualate, L-2-amino-4-phosphonobutyrate (AP4 or APB) and N-methyl-D-aspartate (NMDA) 1°'23. While E A A participate in normal synaptic transmission, under certain circumstances overactivation of their receptors contributes to a variety of pathological processes. The N M D A type of receptor appears to contribute (most) importantly to neuronal damage resulting from ischemiahypoxia 13,26,29, epilepsy s,25, hypoglycemia31,32, trauma 9, and neurodegenerative disorders and dementia ~2,22"36. Despite rapid progress in characterizing both physiological and pathological aspects of N M D A receptor function, the sequence of events linking excessive E A A receptor activation to neurotoxicity remains unclear (see refs. 24, 33, 35 for review). The entry of extracellular Ca 2+ into neuronal cytosol through channels associated with the N M D A receptor appears to play a central role 6"14. This involvement of a receptor-coupled divalent cation channel also implicates (a) Ca2+-activated intracellular second messenger(s) in the events leading to irreversible neuronal injury. Of particular interest in this regard is the possibility of deleterious effects arising from Ca2+-sensitive proteins already known to function beneficially in the N M D A receptor-mediated process of long

term potentiation (LTP): calpain(s), protein kinase C, and calmodulin-dependent protein kinase II. Through the use of inhibitors, considerable progress has been made in documenting the participation of these effector proteins in LTP, although chemical and structural homology among them reduces the selectivity of the probes 4'16. In the present study we examined the role of N M D A receptor- and Ca2+-mediated events during ischemia/ recovery simulated in hippocampal slices from rat brain. We used tissue slices rather than isolated cells to approximate the whole brain better. We used in vitro ischemia rather than exogenous N M D A receptor agonist in order not to distort the proportions of ischemiatriggered events. At the same time, an in vitro approach insured that levels of substrates for cellular energy metabolism (glucose and oxygen) were uniform and easily controlled during both ischemia and recovery and that the tissue was readily accessible for biochemical measurements. We used ATP levels both as an index of short-term ischemia and as a measure of irreversible tissue injury at longer time intervals 7'15. We first defined the model by establishing the dependence of measured ATP on oxygen, glucose and/or Mg 2÷ levels at various stages of ischemia and recovery; then we examined the effects of antagonists of N M D A receptors and Ca 2+dependent intracellular mediator proteins on cellular ATP levels.

Correspondence: R. Pohorecki, Department of Anesthesiology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198 U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

134 ATP, ADP, AMP and calmidazolium were purchased from Sigma Chemical Co. (St. Louis, MO). Racemic ketamine was obtained from Research Biochemicals International (Natick, MA). MK-801 was a generous gift from Merck Sharp Dohme, Inc. (Rahway, N J). All other reagents were of analytical grade. Sprague-Dawley rats weighing 200-250 g were killed by decapitation and their brains quickly removed and placed in ice-cold oxygenated buffer. The hippocampi were dissected as rapidly as possible and transverse slices 400 ~m thick prepared using a McIlwain tissue chopper. The slices were randomly placed in one of two incubation chambers filled with buffer continuously equilibrated with 02/C02 (95/5) at 35 °C. The composition of the buffer was as follows (mM): NaC1 124, KC1 3.1, NaHCO 3 17, CaC12 1.3, MgSO 4 1.3, KH2PO 4 1.3, glucose 10, pH 7.35 at 35 °C. After 120 min of initial incubation, the buffer in one of the two chambers was rapidly replaced with another of the same composition except that glucose was omitted and equilibration was carried out with N 2 / C O 2 (95/5). These in vitro ischemic conditions were maintained for 20 min, at which time the pre-ischemic conditions were restored. The incubation was continued for an additional 240 min, for a total of 380 min. The second chamber served as a sham-ischemic control--i.e., glucose was not omitted in the replacement buffer, and 02/C02 was used for equilibration throughout the entire incubation period. In some experiments the buffer was modified as indicated later. In such cases osmolarity was kept constant by adjusting the NaC1 content. Drugs, when added, were present throughout the entire incubation period. At times indicated under Results, slices were removed from the chambers, rapidly frozen in liquid nitrogen and homogenized in 0.5 N HCIO 4. Each sample was adjusted to pH 6-7 with 0.5 N KOH, cooled to 0 °C and centrifuged to remove excess KCIO4, and passed through a nylon filter (0.4 #m, MSI, Westboro, MA). ATP was determined by HPLC using isocratic elution. The mobile phase included 0.1 M phosphate buffer, 8 mM tetrabutylammonium hydrogen sulfate and 10% acetonitrile (pH 6.0), at a flow rate of 0.6 ml/min. A 15-cm C18 reverse phase silica column, 4-#m particle size (Nova-Pak #86344, Waters, Milford, MA) was used. An external standard was used for quantitation. Protein was determined by a dye binding method (Bio-Rad Protein Assay Kit, Richmond, CA). Each experiment was repeated 5 times and the results presented as mean + S.D. of ATP levels or as a fraction of the corresponding pre-ischemic or pre-anoxic value. Statistical significance was ascertained by ANOVA, using repeated measures when possible, followed by Sheffe's test. The average protein content was 2.01 mg + 0.18 per

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Ischemic brain injury in vitro: protective effects of NMDA receptor antagonists and calmidazolium.

Excessive Ca2+ influx through NMDA receptor-coupled channels has been linked to neuronal cell death. Using an in vitro model of transient brain ischem...
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