Brain Research, 506 (1990) 339-342 Elsevier

339

BRES 23898

Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen-glucose deprivation neuronal injury in cortical cultures Rona G. Giffard 1, Hannelore Monyer 2, Chadwick W. Christine 2 and Dennis W. Choi 2 Departments of 1Anesthesia and 2Neurology, Stanford University Medical center. Stanford, CA 94305 (U.S.A. )

(Accepted 19 September 1989) Key words: Ischemia; Cell death; Glutamate: N-Methyl-D-aspartate; Stroke; Acidosis

The acidosis which accompanies cerebral ischemia in vivo has been thought to contribute to subsequent neuronal injury. However, recent electrophysiological recordings from hippocampal neurons suggest that H + can attenuate N-methyl-D-aspartate (NMDA) receptor-mediated cation influx, likely a key event in the pathogenesis of ischemic neuronal injury. Here we report that moderate extracellular acidosis (pH 6.5) markedly reduced the inward whole cell current induced by NMDA on cultured cortical neurons; at pH 6.1, kainate-induced current was additionally reduced. Furthermore, such acidosis reduced the cortical neuronal injury caused by toxic glutamate exposure, as well as the neuronal degeneration and accumulation of 45Ca 2+ induced by combined oxygen and glucose deprivation. These findings raise the possibility that moderate acidosis may decrease cortical neuronal vulnerability to ischemic damage.

The acidosis which accompanies cerebral ischemia in vivo has been viewed as a possible contributing factor in the pathogenesis of ischemic neuronal damage 26'27. This view had received primary support from observations that preischemic glucose administration worsens both ischemic lactic acidosis and subsequent brain damage is' 21,22,25,30. However, while these observations establish a clear association between acidosis and injury, they do not establish direct causality at the parenchymal level. In counterpoint, recovery of neuronal function in hippocampal slices subjected to hypoxia was actually improved by addition of 10 mM lactic acid to the perfusate, sufficient to reduce pH to 6.8-6.9 (ref. 24). More specific reason to question a causal link between acidosis and ischemic brain injury was provided by a recent electrophysiologic study by Morad and colleagues ~v, who found that moderate acidity (pH 6.6) could markedly reduce N-methyl-D-aspartate ( N M D A ) receptor-activated currents in cultured hippocampal neurons. Since excessive activation of neuronal N M D A receptors by endogenous glutamate has been postulated to be an important factor in hypoxic-ischemic neuronal damage 7232~, we set out to test the hypothesis that moderate extracellular acidosis might protect central neurons against damage by either glutamate exposure, or combined hypoxia and glucose deprivation. We carried out the investigation in cortical culture, where the direct actions of H + on cortical cells could be isolated from the

metabolic and circulatory alterations associated with systemic acidosis in vivo. Experiments were performed in primary cortical cell cultures isolated from fetal mice of 15-17 days gestation as previously described 3. Cortices were dissociated and plated as single cells suspensions on Primaria 24-well culture plates in Eagle's Minimal Essential Medium (MEM, Earle's salts), containing freshly added glutamine (2 mM), and supplemented with 10% horse serum, 10% fetal bovine serum, and glucose (final 21 mM). Cultures were maintained in a 5% CO 2 humidified atmosphere at 37 °C. Glial cell replication was inhibited after 9-11 days in vitro by 1-3 days exposure to 10 5 M cytosine arabinoside. Cells were grown thereafter in a growth medium identical to the plating medium but lacking fetal serum. We tested the effect of H + on m e m b r a n e currents induced by the application of N M D A , recording from single neurons with patch clamp technique in the whole cell configuration w (Fig. la). M g 2+ w a s omitted from the perfusion solutions to unmask N M D A currents at negative potentials 16"2°. The whole cell current induced by perfusion with 10¢tM N M D A at pH 7.3 was progressively attenuated with lowered pH in the range 6.9-6.1 (Fig. lb). Of note, an initial transient current was observed with perfusion of acidic solutions that appeared to be largely an effect of the acidity alone (Fig. la), perhaps due to direct activation of N M D A receptor-linked chan-

Correspondence: D.W. Choi, Department of Neurology, Stanford University Medical Center, Stanford, CA 94305, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

341) nels by extracellular H + (ref. 9). A t p H 6.5, responses to N M D A were reduced much m o r e than responses to kainate, although at p H 6.1, the k a i n a t e - i n d u c e d whole cell current was also substantially r e d u c e d (Fig. l b ) . E x a m i n a t i o n of the N M D A - i n d u c e d whole cell c u r r e n t - v o l t a g e relationship over the range - 8 0 m V to +30 m V suggested little or no voltage d e p e n d e n c e of the block by H + (Fig. lc), The ability of H ÷ to reduce N M D A r e c e p t o r - m e d i a t e d cation influx suggested that it might protect neurons against g l u t a m a t e - i n d u c e d injury. Cortical cultures were e x p o s e d to 500/~M g l u t a m a t e for 5 min, an exposure which destroys a m a j o r i t y of the neuronal population without gross glial d a m a g e 3. The extent of neuronal destruction was q u a n t i t a t e d by m e a s u r e m e n t of lactate d e h y d r o g e n a s e ( L D H ) efflux into the bathing medium the following day le. The glutamate exposure was carried out at chosen p H in a balanced salt solution (BSS) containing (in m M ) NaC1 116, KC1 5.4, MgSO 4 0.8, N a H 2 P O 4 1, CaC12 0.9, glucose 5.5, P I P E S 20 and phenol

NMDA 7.3

a

NMDA 6.5

NMDA 7.3

red I0 mg/liter. The N a H C O 3 concentration was wined as a p p r o p r i a t e for the chosen p H in a 5% CO: atmosphere at 37 °C 4. The exposure was terminated by replacing the exposure solution with M E M . G l u t a m a t e toxicity was highly sensitive to [H*]o. Some suggestion of injury amelioration was seen at pH 6.9; at pH 6.4, injury was r e d u c e d to near zero (Fig. 2). Control experiments showed that 5-min e x p o s u r e to these levels of acidity or to the P I P E S buffer itself did not damage cortical cells in the absence of glutamate. The ability of H ÷ to a t t e n u a t e glutamate neurotoxicity suggests that it might also reduce the ischemia-induced 45Ca2+ accumulation. This 45Ca2~ accumulation correlates well with the extent of subsequent neuronal degeneration 5. For these exposures, the culture m e d i u m was exchanged for d e o x y g e n a t e d BSS lacking glucose, but containing a trace amount of 45Ca2+. Cultures were incubated in an anoxia c h a m b e r (oxygen tension < 0.2% m o n i t o r e d with an oxygen e l e c t r o d e ) at 37 ° and 5% CO2 for 45-60 min. I m m e d i a t e l y after this exposure, the

NMDA 6.1

NMDA 7.,3

NMDA 7.3

6.1

.2 nA 3 0 sec

Koin 7.3

Koin 6.1

Koin 7.,3

11

I I

I 1

Koin 6.9

Koin 7.3

1 nA 30 sec

b 100

~

10 ~ NMDA

I

40

~

C

I00~

Kmnote

t~

-5 ,b 8u "6

-100-

50

OpH73 • pH 6,5

-200

a

-300 -

6.9

6.5

400 -100

-80

-GO

-40

pH6.1

-20

20

4JO....

6.1 Membrone

potentiol (mY)

Fig. 1. Acidic solutions reduce NMDA-induced whole cell currents, a: representative tracings demonstrate the ability of H ~ to reduce NMDAand kainate-induced whole cell currents. Arrows indicate onset of perfusion of the indicated solutions, from perfusion pipettes within 100/am of the cell body. Holding potential was -60 mV in both examples. Note that application of acidic solutions (both with and without added agonist) elicited an initial transient current. The pipette solution was (in mM): CsCI 140, HEPES 10, CaCI 2 0.5 and EGTA 5. Perfusion solutions contained (in mM): NaCI 125, KCI 2.8, CaCI 2 1, PIPES 10 and 1 /aM tetrodotoxin; 5/aM glycine was included in experiments where NMDA-induced currents were measured, b: compiled results of several experiments similar to those in (a). Whole cell currents at the indicated pH are expressed as a percentage of the control response at pH 7.3; bars indicate S.E.M. Initial transient currents were ignored. All tested conditions produced responses significantly less than that produced by the pH 7.3 control except kainate at 6.9 (P < 0.0t, two-tailed t-test). c: current-voltage relationship for NMDA-induced responses at various pH on a single cell, representative of results obtained on 5 other cells. Current values represent the NMDA-elicited current (leak current at the appropriate voltage has been subtracted). Conductance values from linear regression were: at pH 7.3, 4.41 nS; pH 6.5, 1.54 nS; and pH 6.1, 0.39 nS.

341

100-

i6oo

a

~20

80-

~E 1200J

100 ~ 80

~ 800

~" 60

b

60.

i

40. 20.

7.4

6.9

**

T

40

4001

20

0 -6.4

Fig. 2. Protective effect of extracellular acidity upon glutamate neurotoxicity. Cultured neoeortical ceils were exposed to 500/~M glutamate for 5 min at the indicated pH, and LDH was measured in the bathing medium 18 h later (mean _+ S.E.M., n = 5), scaled to the mean amount seen in the cultures exposed at pH 7.4 (= 100). The background level of LDH found in cultures exposed to sham wash (similar at either pH 7.4 or pH 6.4) has been subtracted from all values. * and ** indicate significant difference from injury at pH 7.4 (P < 0.05 and P < 0.01, respectively, by two-tailed t-test and Bonferroni correction for two comparisons.

exposure solution was r e m o v e d and the cells were washed three times before being solubilized with 0.2% SDS at 37 °C. Net cellular 45Ca2+ accumulation was d e t e r m i n e d with a scintillation counter. C o n s i d e r a b l e 45Ca2+ accumulation was evident after ischemic exposure at p H 7.4; lowering the p H to 6.4 m a r k e d l y r e d u c e d this accumulation (Fig. 3a). This protection was c o m p a r a b l e in extent to that p r o d u c e d by the N M D A antagonist, d e x t r o r p h a n 5. In o t h e r e x p e r i m e n t s , 45Ca 2+ was not a d d e d and after terminating the ischemic exposure by exchanging the m e d i u m for o x y g e n a t e d M E M , the cultures were ret u r n e d to the n o r m a l culture incubator. The day following such ischemic exposure at p H 7.4, widespread neuronal d e g e n e r a t i o n and substantial L D H effiux occurred 6 (Fig. 3b). H o w e v e r , if p H was d r o p p e d to 6.4, on the following day there was a substantial reduction in both the m o r p h o l o g i c evidence of neuronal d a m a g e and L D H effiux (Fig. 3b). Similar exposure to p H 6.4 in the presence of oxygen and glucose did not d a m a g e cortical cells. These e x p e r i m e n t s d e m o n s t r a t e a protective effect of m o d e r a t e extracellular acidosis on cultured neocortical neurons, e x p o s e d either to c o m b i n e d oxygen and glucose d e p r i v a t i o n , or to exogenously applied glutamate. Neur o p r o t e c t i o n was seen at levels of extracellular acidosis d e m o n s t r a t e d to occur during in vivo ischemia 11'19'25'26. While further study will be n e e d e d to establish the exact basis for this neuroprotective action, it is most likely accounted for by a reduction in the neuronal injury caused by excess exposure to e n d o g e n o u s excitatory amino acids released during ischemia. In a g r e e m e n t with the study of M o r a d et al. 17 on

0 -7.4

6.4

7.4

6.4

Fig. 3. Protective effect of extracellular acidity on ischemic injury in vitro, a: 45Ca2+ influx into cells exposed to ischemia for 45 min. Each bar depicts the mean counts accumulated by 4 sister cultures + S.E.M., exposed to ischemia at pH 7.4 or pH 6.4. Background counts retained by cultures exposed to sham wash were subtracted from the values shown, b: LDH release from cells exposed to ischemia for 50 rain. Each bar depicts the mean of 4 sister cultures + S.E.M., scaled to the mean release seen at pH 7.4 (= 100). Background LDH released by cultures exposed to sham wash was subtracted from the values shown. For both panels, ** indicates significant difference from the pH 7.4 condition (P < 0.01 by two-tailed t-test).

h i p p o c a m p a l neurons, we found that lowering extraceliular p H sharply r e d u c e d the whole cell current associated with activation of cortical neuronal N M D A receptors. Not only was this reduction p r o f o u n d , it was also i n d e p e n d e n t of m e m b r a n e potential, a feature which could allow the n e u r o p r o t e c t i v e effect of H + to persist under conditions of ischemic depolarization. This voltage i n d e p e n d e n c e may be most consistent with a site of H + action near the external m e m b r a n e face. Selective blockade of N M D A receptors is a sufficient explanation for the observed ability of H + to a t t e n u a t e glutamate neurotoxicity, as well as the 45Ca2+ and neuronal injury induced by ischemic exposure. We have previously found that selective N M D A antagonists could block both glutamate neurotoxicity 2 and hypoxic neuronal injury v in cortical cultures, consistent with the idea that N M D A r e c e p t o r activation is critical to the toxic Ca 2+ influx that may underlie both events 1"]5. H o w e v e r , it is possible that attenuation of n o n - N M D A r e c e p t o r activation might also contribute to the observed effects of H +. That extracellular acidosis can destroy brain cells is undisputed. Kraig et al. o b s e r v e d coagulative necrosis of brains injected with enough lactic acid to d r o p extracellular p H below 5.3 (ref. 13). It is likely that H ÷ can trigger a n u m b e r of potentially cytotoxic events, including e n h a n c e m e n t of free radical formation~7; glia in particular may be vulnerable to H + - i n d u c e d d a m a g e 8"14. F u r t h e r m o r e , our observations do not address the possibility that brain acidosis may have i m p o r t a n t indirect deleterious effects in vivo, for e x a m p l e m e d i a t e d by unfavorable alterations in b l o o d flow. H o w e v e r , the results r e p o r t e d here suggest specifically that m o d e r a t e

342 levels of e x t r a c e l l u l a r acidosis could also h a v e an i m p o r -

r o p r o t e c t i v e effect of acidosis d e s c r i b e d h e r e e x t e n d s ais~

tant n e u r o p r o t e c t i v e effect in i s c h e m i a , directly r e d u c i n g

to h i p p o c a m p a l n e u r o n s .

neuronal

vulnerability

m e d i a t e d by N M D A

to

that

component

of

injury

receptors.

A s this r e p o r t was b e i n g p r e p a r e d , we b e c a m e a w a r e of c o m p l e m e n t a r y o b s e r v a t i o n s i n d e p e n d e n t l y m a d e by T o m b a u g h and S a p o l s k y 29, which suggest that the neu-

1 Choi, D.W., Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends" Neurosci., 11 (1988) 465-469. 2 Choi, D.W., Koh, J. and Peters, S., Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists, J. Neurosci., 8 (1988) 185-196. 3 Choi, D.W., Maulucci-Gedde, M. and Kriegstein, A.R., Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. 4 Dawson, R M . C . , Elliot, D.C., Elliot, W.H. and Jones, K.M., Data for Biochemical Research, 3rd edn, Oxford Science Publications, Oxford, 1986, p. 433. 5 Goldberg, M.P., Kurth, M.C., Giffard, R.G. and Choi, D.W., 45Calcium accumulation and intraceUular calcium during in vitro qschemia', Soc. Neurosci. Abstr., 15 (1989) 803. 6 Goldberg, M.P., Monyer, H. and Choi, D.W., Cortical neuronal injury in vitro following combined glucose and oxygen deprivation: ionic dependence and delayed protection by NMDA antagonists, Soc. Neurosci. Abstr., 14 (1988) 745. 7 Goldberg, M.E, Weiss, J.W., Pham, P.C. and Choi, D.W., N-methyl-D-aspartate receptors mediate hypoxic neuronal injury in cortical culture. J. Pharmacol. Exp. Ther., 243 (1987) 784-791. 8 Goldman, S.A., Pulsinetli, W., Kraig, R.P. and Plum, F., Tolerance of neurons and glia to acid exposure in vitro, Soc. Neurosci. Abstr., 12 (1986) 65. 9 Grantyn, R. and Lux, H.D., Similarity and mutual exclusion of NMDA- and proton-activated transient Na + currents in rat tectal neurons, Neurosci. Lett., 89 (1988) 198-203. 10 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, EJ., Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 11 Harris, R J . , Richards, P.G., Symon, L., Habib, A.-H.A. and Rosenstein, J., pH, K + and PO 2 of the extracellular space during ischaemia of primate cerebral cortex, J. Cereb. Blood Flow Metab., 7 (1987) 599-604. 12 Koh, J. and Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase effiux assay, J. Neurosci. Methods, 20 (1987) 83-90. 13 Kraig, R.E, Petito, C.K., Plum, E and Pulsinelli, W.A., Hydrogen ions kill brain at concentrations reached in ischemia, J. Cereb. Blood Flow Metab., 7 (1987) 379-386. 14 Kraig, R.P., Pulsinelli, W.A. and Plum, E, Hydrogen ion buffering during complete brain ischemia, Brain Research, 342 (1985) 281-290. 15 MacDermon, A.B. and Dale, N., Receptors, ion channels and

We thank M. Kurth for assistance with tile ~5Ca:~ paradigm, M.P. Goldberg for assistance with the ischemia paradigm, and K. Rose for cell culture assistance. This research was supported by NIH Grants NS 12151 and NS26907. H.M. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.

16

17

18 19

20

21

22

23

24

25

26

27 28

29

30

synaptic potentials underlying the integrative actions of excitatory amino acids, Trends Neurosci., 10 (1987) 280-284. Mayer, M.L., Westbrook, G.L and Guthrie, P.B., Voltagedependent block by Mg2+ of NMDA responses in spinal cord neurones, Nature (Lond.), 309 (1984) 261-263. Morad, M., Dichter, M. and Tang, C.M., The NMDA activated current in hippocampal neurons is highly sensitive to [H+]o, Soc. Neurosci. Abstr., 14 (1988) 791. Myers, R.E. and Yamaguchi, S., Nervous system effects of cardiac arrest in monkeys, Arch. Neurol., 34 (1977) 65-74. Nemoto, E.M. and Frinak, S., Brain tissue pH after global brain ischemia and barbiturate loading in rats, Stroke, 12 (1981) 77-82. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurons, Nature (Lond.), 307 (1984) 462-465. Pulsinelli, W.A., Waldman, S., Rawlinson, D. and Plum, E, Moderate hyperglycemia augments ischemic brain damage: a neuropathologic study in the rat, Neurology, 32 (1982) 12391246. Rehncrona, S., Rosen, I. and Siesj6, B.K., Brain lactic acidosis and ischemic cell damage. I. Biochemistry and neurophysiology, J. Cereb. Blood Flow Metab., 1 (1981) 297-33l. Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypozic/ischemic brain damage, Ann. Neurol.. 19 (1986) 105-111. Schurr, A., Dong, W.Q., Reid, K.H., West, C.A. and Rigor, B.M., Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro, Brain Research, 438 (1988) 311-314. Siemkowicz, E. and Hansen, A.J., Brain extracellular ion composition and EEG activity following 10 minutes ischemia in normo- and hyperglycemic rats, Stroke, 12 (1981) 236-240. Siesj6, B.K., Acid-base homeostasis in the brain: physiology, chemistry and neurochemical pathology, Prog. Brain Res., 63 (1985) 121-154. Siesj6, B.K., Mechanisms of ischemic brain damage, Crit. Care Med., 16 (1988) 954-963. Simon, R.P., Swan, J.H., Griffiths, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. Tombaugh, G.C. and Sapolsky, R.M., Mild acidosis protects hippocampal neurons from injury induced by oxygen and glucose deprivation, Brain Research, 506 (1990) 343-345. Welsh, E A . , Ginsberg, M.D., Rieder, W. and Budd, W.W., Deleterious effects of glucose pretreatment on recovery from diffuse cerebral ischemia in the cat. It. Regional metabolite levels, Stroke, 11 (1980) 355-363.