Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro

Neuroscience Vol. 68, No. 3, pp. 615-619, 1995

Pergamon

0306-4522(95)00232-4

Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00

Zetter to Neuroscience BLOCKADE OF GLUTAMATE RECEPTORS UNMASKS NEURONAL APOPTOSIS AFTER OXYGEN-GLUCOSE DEPRIVATION I N VITRO B. J. G W A G , D. L O B N E R , J. Y. K O H , M. B. W I E and D. W. C H O I * Department of Neurology and Center for the Study of Nervous System Injury, Box 8111, Washington University School of Medicine, St Louis, MO 63110, U.S.A.

Mouse cortical cell cultures exposed to transient oxygen-glucose deprivation developed marked acute cell body swelling followed by neurodegeneration, consistent with necrosis-type death. This death was not attenuated by the protein synthesis inhibitor, cycloheximide, but was attenuated by addition of the N-methyI-D-asparate antagonist, MK-801 (dizocUpine maleate), and the ~-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid/kainate antagonist, 6-cyano-7nitroquinoxaline-2,3-dione. If the deprivation insult was extended to overcome the protective effect of glutamate antagonists, neuronal death resulted that was associated with cell body shrinkage and DNA fragmentation, and was attenuated by cycloheximide. These data suggest that oxygen-glucose deprivation can induce in cortical neurons both excitotoxic necrosis, and apoptosis dependent on new macromolecule synthesis.

calcium ion entry, accompanied by chloride and water, leading to marked early intracellular volume expansion. 6'26'29At least in our murine cortical culture system, rapidly triggered, N M D A receptor-mediated excitotoxicity is associated with diffuse D N A degradation, and is not sensitive to protein synthesis inhibitors. 8'2° In addition, addition of cycloheximide does not reduce the neuronal death induced by 24-h exposure to e-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( A M P A ) or kainate (B. J. Gwag and D. Choi, unpublished results). Neither cycloheximide sensitivity nor laddered D N A fragmentation

N o r m a l programmed death of neurons in the developing nervous system generally takes place by apoptosis, a type of cell death characterized by cell body shrinkage, nuclear condensation, and discrete D N A fragmentation) 6'~8'~9 In addition, apoptosis in many cases appears to require new protein synthesis. 24'37 In contrast, pathological neuronal death induced by several environmental insults, including hypoxia--ischemia, generally occurs by necrosis, a type of cell death characterized by early prominent cell body swellingJ 2'j3'19 The implication of excitotoxicity as an important component of ischemic neuronal death 3'7"33has added specific support to this distinction. Overstimulation of neuronal N M D A and A M P A / k a i n a t e classes of glutamate receptors permits excessive sodium and

CTRL CHX MK-801

*To whom correspondence should be addressed. AMPA, c~-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EDTA, ethylenediaminetetraacetic acid; NMDA, N-methyl-D-aspartate; LDH, lactate dehydrogenase; TUNEL, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.

Abbreviations:

Table 1. Brief deprivation of oxygen and glucose produces NMDA receptor-mediated excitotoxic neuronal death LDH release Treatment

24 h

48 h

60 ___7 62 _ 7 11 + 3*

64 __+6 63 __+7 17 + 3*

Cortical cultures were exposed to oxygen-glucose deprivation for 50min, alone (CTRL), or in the presence of l#g/ml cycloheximide (CHX) or 10~M MK-801 (dizocilpine maleate). LDH in the bathing medium was measured 24 and 48h later, mean + S.E.M. (n = 12 cultures per condition). *Significant difference from relevant control at P < 0.05 using analysis of variance and Student-Neuman Keuls test. Mouse cortical cells prepared from embryonic day 15-16 fetal mice were plated on a glia monolayer and cultured for 14-16 daysfl 8 Animals were handled in accordance with a protocol approved by our institutional animal care committee. Plating density was approximately 2.5 × 105 cells per well. Cortical cell cultures containing both neurons and glia, 14-16 days in vitro, were deprived of oxygen and glucose by placing the cultures in a hypoxia chamber and exchanging media with oxygen-depleted, glucose-free balanced salt solution (BSS0) (mM): NaCI 116, KC1 5.4, MgSO 4 0.8, NaH2PO 4 1, CaCI: 0.9, and Phenol Red 10mg/l. The deprivation was terminated by washout of the exposure medium with oxygenated balanced salt solution containing 5.5 mM glucose. Per cent neuronal death was scaled to the value measured at 24 and 48 h in sister cultures exposed to 500/zM NMDA (= I00), a treatment which produced nearly complete neuronal death. 615

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Fig. 1. Latent neuronal degeneration following prolonged oxygen-glucose deprivation in the presence of MK-801 and CNQX. (A) Phase-contrast photomicrographs of a cortical cell culture immediately after 90 min exposure to oxygen-glucose deprivation. (B) Same as A, but with 10 #M MK-801 and 100/~M CNQX added during oxygen-glucose deprivation. (C, D) Same as B, but 24 (C) or 48 h (D) later. Scale bar = 50 t~m. Note the gradual degeneration of the same cortical neurons (B-D) following the deprivation in the presence of glutamate antagonists. was detected in cerebellar cultures exposed to glutamate toxicity.9 Recently, however, the notion that ischemic neuronal death occurs strictly by necrosis has been challenged. Characteristics of apoptosis such as internucleosomal D N A fragmentation or sensitivity to protein synthesis inhibitors have been found in several animal models of ischemic brain injury. 22'23"25'3° In previous studies of oxygen-glucose deprivationinduced injury in cortical cell cultures, we have observed excitotoxic necrosis without evidence of apoptosis. 12'13Here, we considered the specific possibility that fulminant excitotoxic necrosis might mask a more slowly evolving apoptosis death. TM Deprivation of oxygen and glucose for 45-55 min induced immediate neuronal swelling, followed by neuronal degeneration over the next 24h, accompanied by release of lactate dehydrogenase (LDH) into the bathing media. Glia remained intact, and the levels of LDH in the media remained stable between 24 and 48h (Table 1). This neuronal degeneration was attenuated by addition of 10/~M MK-801 during the deprivation period, but not by addition of 1 ~tg/ml cycloheximide during and after the deprivation (Table 1). As previously reported, combined blockade of N M D A and AMPA/kainate receptor-mediated excitotoxicity, using 10~M MK-801 and 100/~M

6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), resulted in additive neuroprotection, such that 90-100min of oxygen glucose deprivation only induced limited neuronal death after 24 h.17 However, if the cultures were followed by another day, many neurons went on to develop cell body shrinkage, neurite degeneration, and eventual lysis (Fig. 1, Table 2). Soluble D N A extracted at the 24-h timepoint revealed fragmentation into multiples of 200 bp (Fig. 2A), typical of apoptosis. 15'16'19 In situ end labelling of D N A with the T U N E L stain indicated that DNA fragmentation was largely confined to neurons (Fig. 2B). D N A laddering was barely detectable in cultures partially damaged by 50 min exposure to oxygen-glucose deprivation in the absence of glutamate antagonist drugs (Fig. 2A). In several neuronal and non-neuronal systems, apoptosis can be blocked by inhibitors of transcription and translationfl4"37 Indeed, neuronal death in our cortical cultures induced by serum deprivation can be blocked by addition of l/~g/ml cycloheximide. 21 Sister cultures treated with 1/~g/ml cycloheximide showed significant reduction in protein synthesis (3H-lysine incorporation into protein) without intrinsic toxicity (unpublished data). Cycloheximide addition failed to attenuate the neuronal death induced by 50 rain oxygen-glucose deprivation (Table 1), but did attenuate neuronal death in

Apoptosis unmasked after blockade of excitotoxicity cultures exposed to 9 0 - 1 0 0 m i n oxygen-glucose d e p r i v a t i o n in the presence o f MK-801 a n d C N Q X (Table 2). In particular, the latent d e a t h emerging between 24 a n d 48 h after the insult in this p a r a d i g m was m a r k e d l y a t t e n u a t e d (Table 2). These results suggest t h a t a single oxygen-glucose d e p r i v a t i o n insult can induce in cortical n e u r o n s b o t h excitotoxic necrosis, a n d apoptosis d e p e n d e n t on new protein synthesis. While there are presently no absolute (necessary or sufficient) criteria for defining apoptosis, the c o m b i n a t i o n o f m o r p h o l o g y , internucleosomal D N A f r a g m e n t a t i o n , a n d sensitivity to cycloheximide observed here goes b e y o n d t h a t previously reported in ischemia models to date, 22"23"25"3° a n d provides a reasonable case suitable for future refinement. Consistent with the original thinking o f K e r r et al. 19 w h o considered h y p o x i c - ischemic n e u r o n a l d e a t h to be a defining example of necrosis, excitotoxic necrosis was the d o m i n a n t injury u n d e r untreated conditions. However, ifexcitotoxic necrosis was blocked pharmacologically, then a more indolent, delayed d e a t h - - l i k e l y a p o p t o s i s - - w a s revealed.

A

617 a

b

c

bp 1018 50~ 39~ 298 220 - -

Table 2. Cycloheximide attenuates neuronal death in cultures exposed to prolonged oxygen-glucose deprivation in the presence of glutamate antagonists (a) Treatment CTRL MK-801/CNQX MK-801/CNQX/CHX (b) Treatment MK-801/CNQX MK-801/CNQX/CHX

24 h

LDH release 48 h

100 + 5 20 + 3* 10 + 2*¢

105 + 7 61 _ 4* 19 + 3*t

LDH release vs death by cell counts at 48 h LDH assay Cell counts 46 + 8 15 + 3*

43 + 3 11 + 2*

(a) Cultures were exposed to oxygen-glucose deprivation for 90 100min alone (CTRL), in the presence of 10/~M MK-801/100,uM CNQX (MK-801/CNQX), or in the presence of 10pM MK-801/100pM CNQX/I/tg/ml cycloheximide (MK-801/CNQX/CHX). LDH in the bathing medium was measured 24 and 48 h later, mean ±S.E.M. (n = 12 cultures per condition), scaled to the mean value ( = I00) corresponding to the near-complete neuronal death induced in sister cultures by exposure to 500/tM NMDA. *Significant difference from relevant control; tsignificant difference between corresponding CHX added and CHX absent conditions, at P < 0.05 using analysis of variance and Student Neuman-Keuls test. (b) Cell counts of dead cells labeled by Trypan Blue dye confirmed results obtained by LDH assay. Cultures were exposed to oxygen-glucose deprivation for 90 rain in the presence of 10/~M MK-801/100/~M CNQX, with or without 1/~g/ml cycloheximide. LDH in the bathing medium was measured 48 h later, mean + S.E.M. (n = 0.8 cultures per condition), scaled to the mean LDH value corresponding to the near-complete neuronal death induced by 500/IM NMDA in sister cultures. Immediately after LDH assay, cultures were exposed to Trypan Blue dye and per cent neuronal death was assessed by counting stained neurons, mean + S.E.M. (n = 20 random fields in four cultures per condition). * Indicates significant difference between relevant CHX added and CHX absent conditions, at P < 0.05 using analysis of variance and Student Neuman Keuls' test.

Fig. 2. DNA fragmentation after oxygen~lucose deprivation. (A) Agarose gel electrophoresis of DNA extracted from sister cultures (four per lane) exposed 24 h previously to sham wash (a, lane 1), oxygen-glucose deprivation for 50 rain alone (b, lane 2) or oxygen-glucose deprivation for 90min in the presence of 10#M MK-801 and 100/~M CNQX (c, lane 3). (B) Brightfield photomicrographs of cultures exposed 24h previously to sham wash (BI) or oxygen-glucose deprivation for 90 min in the presence of 10#M MK-801 and 100/tM CNQX (B2). In situ DNA end-labeling was carried out using biotin-16-dUTP and terminal deoxynucleotidyl transferase, Soluble DNA to identify DNA fragments was prepared as described, t5 Briefly, cultures were washed in phosphate-buffered saline and incubated for 30~0 min at 4°C in 100 #1 of lysis buffer: 0.5% Triton X-100 (Sigma), 5 mM Tris pH 7.4, and 20 mM EDTA. The cell lysates from four wells for each group were combined into an Eppendorf tube and centrifuged at 4°C for 15 min. The supernatant was extracted with phenol:chloroform :isoamylalcohol (25 : 24: 1), precipitated in ethanol, and then resuspended in dH20 and electrophoresed in 1.5% agarose gel containing 300 ng/ml ethidium bromide. After electrophoresis, the gel was incubated in DNAse-free RNAse solution for 2 h. A 1 kb DNA ladder (Gibco BRL) was used as standard. To detect in situ DNA fragmentationJ t cultures were fixed in 4% paraformaldehyde overnight at 4~'C, incubated in 20 mg/ml proteinase K (Sigma) at room temperature for 5 min, and treated with 2% H202 for 2 min. Each well was then incubated at 3 7 C for l h in 200 #1 of reaction mixture containing 12 ,u M biotin16-dUTP (Boehringer Mannheim Biochemicals), 60U terminal deoxytransferase, 30mM Tris, 140mM sodium cacodylate pH 6.6, and 1 mM cobalt chloride. The biotinylated DNA was linked to extravidin peroxidase (Sigma) and visualized by diaminobenzidine reaction.

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We postulate that this apoptosis death is triggered in parallel with, but masked by, excitotoxic necrosis. We have also observed that young (seven days in vitro) cultured cortical neurons, which express few glutamate receptors, die by apoptosis when confronted with glucose deprivation in the absence of glutamate antagonists (unpublished data). Our results thus support the emerging idea that neuronal apoptosis may occur after ischemic insults in vivo 22,23,25,30and add the notions of parallel occurrence and hierarchy. Present experiments are notable in delineating parallel necrosis and apoptosis induced by a single insult in the same neurons. Further study will be needed to define possible

links between neuronal excitotic necrosis and apoptosis under oxygen-glucose deprivation conditions. Some possible candidates are energy failure, 2'34'36 downregulation of protein kinase activity, ~'4't4 oxidative stress, ~°'27'31 or cytoskeletal damage. 5'3~35 Regardless of the identity of these triggers, our results raise the possibility that the neuroprotective effects of anti-excitotoxic strategies in the setting of stroke or cardiac arrest may be further enhanced by manipulations specifically designed to limit neuronal apoptosis. Acknowledgements--This study was supported by grant

NS 32636 from NINDS (DWC).

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