Neuros'czenee Letter.~, 139 (1992) 191 193 ~" 1992 Elsevier Scientific Pubhshers Ireland Ltd. All rights reserved 0304-3940/92:$ 05.00

191

NSL 08625

The neuro 3rotective effects of dextromethorphan on guinea pig-derived hippocampal slices during hypoxia R i c h a r d J. R a d e k a n d W i l l i a m J. G i a r d i n a Department o/Neuroscwnee, Abbott Laboratories, Abbott Park, IL 60064-3500 ( USA )

Recewed 12 October 1991, Revised version received 15 February 1992: Accepted 24 February 1992) Key words.

Dextromethorphan, AP-5; Hippocampal slice, Hypoxla, Lowered temperature: Population spike

The purpose of this study was to determine whether dextromethorphan, an oplold class antltusswe, prevents hypoxla-mduced loss o1"nerve function m an in vitro hippocampal slice preparation. The evoked population spike (PS) was recorded from CA1 pyramidal cell~ of guinea plg-derwed h~ppocampal slices. Hippocampal slices were superfused with O, (95%)/CO: (5%) gassed artificial cerebral spinal fluid (ACSF) at 37°C The PS did not recover during reoxygenation in shces that were made hypoxic for 30 mm by exposure to N_,(95%)/CO: (5%) gassed ACSF m place of oxygenated ACSF. The PS recovered during reoxygenatlon, following 30 mm of hypoxxa, in 9 of I0 slices treated with dextromethorphan ( 100/aM }and in 4 of 6 slices treated with D,L-2-ammo-5-phosphono-vaterate(AP-5) 1100,uM), an NMDA receptor antagomst. The mean PS amphtudes, one hour after perfuslon with oxygenated ACSF, were 42% and 51%, respectively, of the pre-hypoxla amphtude The PS recovered during reoxygenat~on in all of seven slices superfused with lowered temperature ACSF (25°C} during 30 min of hypoxla. The results show that dextromethorphan, like the NMDA antagonist AP-5 and lowered temperature, protected neurons from hypoxla-induced inJury m the hippocampus

Extracellular concentrations o f the e n d o g e n o u s Nmethyl-D-aspartate ( N M D A ) receptor ligand glutamate are greatly increased during brain ischemia [1]. Ischemia is a condition o f restricted blood flow that creates an hypoxic state in the affected tissue. Excessive levels o f glutamate are neurotoxic [2] and these excesses are believed to be responsible for the neuronal d a m a g e that results from ischemia. D,L-2-Amino-5-phosphono-valerate (AP-5) is a noncompetitive N M D A receptor antagonist which blocks b o t h evoked and s p o n t a n e o u s electrophysiological responses in the h i p p o c a m p u s [5]. AP-5 is one o f several substances that prevents hypoxia-induced loss o f the neurotransmission in the h i p p o c a m p u s [4]. D e x t r o r p h a n , a d e x t r o r o t a r y m o r p h i n a n opioid, and the structurally related d e x t r o m e t h o r p h a n block N M D A evoked responses in brain slices [12] and attenuate glutamate neurotoxicity in cortical cell cultures [3]. These agents also protect cortical cell cultures from the effects o f hypoxia and possess anti-ischemic properties in rabbits [6, 8]. The purpose o f this study was to assess the effect o f d e x t r o m e t h o r p h a n on functional neurotransmission in Correspondence. R.J. Radek, Department of Neuroscience, D-47W,One Abbott Park Road, Abbott Laboratories, Abbott Park, IL 60064-3500, USA.

hypoxic brain tissue and to show its relative efficacy c o m p a r e d to AP-5. In addition, the present study compared the protective effects o f N M D A receptor blockade with that p r o d u c e d by lowered temperature. Hypothermia has been shown to be a highly effective method o f blocking hypoxia-induced loss o f neuronal function in vitro [1 1]. Previous studies o f the cerebroprotective effects o f d e x t r o m e t h o r p h a n have utilized histological techniques to assess hypoxic or neurochemical injury. While such studies show that d e x t r o m e t h o r p h a n - t r e a t e d hypoxic neurons are morphologically intact, they do not provide functional evidence for the extent o f that neuroprotection. The neuroprotective effects o f drugs during hypoxia can be studied in vitro using the hippocampal slice preparation. Hypoxta o f long enough duration will cause the irreversible loss o f the evoked population spike (PS) recorded from the CA1 area of the hippocampus. PS amplitude is a sensitive measure of hypoxic injury, and the h i p p o c a m p u s is responsive to the N M D A class o f pharmacological agents. The h i p p o c a m p u s was rapidly excised from male albino guinea pigs (200 300 g) and cut transversely to the long axis into 400-/~m thick slices. The slices were immediately placed on nylon nets in a recording chamber. While resting on the nets, the upper surfaces o f the slices were gassed with 95% oxygen and 5% carbon dioxide,

1~)2

and the lower surfitces were m contact \~lth oxygenaled artificial cerebrospinal fluid (ACSF). No recordings were attempted during the first hour l\)llowing the cutting procedure. The composition of the A('SF x~as (m mM): NaCI 126.0, KCI 4.0, KH_,PO4 1.4, MgSO4 1.3. NaHCO3 26.0, CaCI2 2.4, glucose 4; pH 7.4. ACSF was gassed with rather O~ 95%/CO~-5% (oxygenated, OA('SF~ or N~ 95%/CO, 5% (N-ACSF). Experiments were begun by submerging the >lices and superfusing them with O-ACSF at 3T~C. ACSF flow rate through the chamber was 4 ml/min. The PS wa,~ evoked through a bipolar nichrome wire-sumulatmg electrode placed in the Shaffer collaterals. Extracellular recordings were made with a thin-walled glass capillary electrode 11 5 M{~ resistance) placed in the stratum pyramidale of CA1. The stimulus strength was adjusted to produce a PS amplitude of 3 4 mV. A stable PS was established during a 15- to 30-mm period belbre data collection began. The PS response was measured from the trough of the initial negativity to the peak of the second positivity (see arrows in Fig. IA, pre-hypoxm), using data acquisition software on a personal computer, The experimental protocol consisted of 5-ram baseline recordings during which slices were superfused with OACSF at 37%'. This was followed by a 10-rain superfuslon ~ith O-ACSF containing one of the dtug,~ or superfusion with lowered temperature (25:C) O-ACSF. The slices were then superfused for an additional 30 mm with either drug-containing N-ACSF or lower-temperature N- ACSF. The oxygen content of the ACSF covering the slices was measured using a Radiometer gas analyzer. The oxygen level dropped fl'om 80% to 17% 10 rain after mitiating N - A C S F flow into the chamber. Alter 20 rain, the oxygen level was down to 4%. After 30 rain of exposure to N-ACSF, the slices were superfused for 10 rain with either O-ACSF containing the drugs or Mw'er-temperature O-ACSF. This period was I\)llowed by 60 min of perfusion with drug-free O-ACSF at 37%'. Exoked responses were recorded every 30 s throughout the experiment. AP-5, dextromethorphan, and chemical reagents for the ACSF were obtamed from Sigma Chelnical Company. N - A C S F superfusion of I 5 rain had little effect on the PS amphtude. The PS began to diminish after 6~-7 rain of hypoxia, and was completely abolished in most slices after 7 8 min of hypoxia. In several sliceb the PS persisted longer, but invariably disappeared after 10 mm of hypoxia. In preliminary experiments, reperfusion with O-ACSF after 10 or 20 min of hypoxla resulted in full recovery of the PS. None of the slices exposed to 30 mm of hypoxia recovered the PS after reoxygenation. Lowered temperature, AP-5 and dextromethorphan reduced the PS amplitude (without hypoxia), but reperfuslon

A ASCF

B ACSF + Dextromethorphan (100 gM)

Pre-Hypoxla

Hypoxia 10 Minutes

l

~

60 Minutes

tqg 1 Effect>of hypoxm with and without superfuslon of dextromethorphan Vemcal bar-2 mV, horizontal bar=2 ms

with drug-free O-ACSF at 37°C resulted in a return of baseline PS amplitudes within 1 h. Measuring the PS 1 h alter reperfusion with drug-free O-ACSF at 37°C in the hypoxia experiments assured that any sustained changes in PS amplitude were only a result of the hypoxia-mduced injury and not any lingering drug effects. Fig. 1 shows typical PS tracings before, during and after hypoxia. Ten minutes of exposure to N-ACSF or N-ACSF with dextromethorphan (100/+M) resulted in a total loss of the PS (Fig. IA,B, hypoxia 10 mm). The slices were exposed to a total of 30 min of hypoxia. No detectable response was evident at 60 min after reperfusion with O-ACSF in untreated slices (Fig. 1A, post-hypoxia 60 min). In contrast, the PS reappeared after reperfusion with O-ACSF in a slice treated with 100/aM dextromethorphan (Fig. 1B, post-hypoxia 60 rain): m the TABLE 1 EFFECTS OF LOWERED TEMPERATURE, AP-5 AND DEXTROMETHORPHAN (DEX) ON THE HYPOXIC HIPPOCAMPUS Treatment

Number of Pre-hypoxla Posl-hypoxla % RccoveD shoes basehne Ih surviving PS amphtude PS amphludc 30-mm hypoxm

N-ACSF

0:10

3 7+_(i 1

CJ

0

N-ACSF

7:7*

3 2+_t).1

2.8 ~0.5

88

Ca 25cC AP-5, lO(}yM 4:6*

3 5~112

I 9~117

51

DEX, 10/tM 1,5 DEX, 100/,tM 9/10"

31 34+0.2

Ill 1.3_+0.3

3~ 42

*t'- 0 1)5 xs N-ACSF. one-tailed Fisher", exacl tc',t

193

example shown, the PS recovered to 51% of baseline amphtude. Table 1 show s a summary of the effects of dextromethorphan, AP-5 and lowered temperature on the hypoxlc hippocampal slice. Data are presented as the number of slices surviving 30 min of hypoxia over the total number of slices recorded for each treatment. A slice was considered to have survived ff a sp~ke at least 20% of baseline could be recorded. Mean PS amplitudes (in mV + S.E.M.) were calculated from those slices that survived hypoxla. Pre-hypoxia PS amphtudes were averaged from the 5-min baseline recordings. Post-hypoxia PS amplitudes were averaged from the 5-min period at 1 h after superfusion w'lth drug-free O-ACSF at 37°C. The PS rapidly declined and wab completely abolished during hypoxia. Superfusion w'ith lowered-temperature NACSF resulted in all of 7 shces surviving with an average recovered PS amplitude of 88%. Superfusion with AP-5 at 100/aM during hypoxla resulted in 4 of 6 slices recovering the PS after reo,~ygenation. The average recovery of the PS in these 4 slices was 51%. When slices were superfused with dextromethorphan at 100/aM, 9 out of 10 slices had a measurable PS, with an average recovery in the 9 shoes o1"42%. Only' one (33% recovery) of 5 slices was protected by 10/aM dextromethorphan. The results show that dextromethorphan reduces hvpoxia-induced injury to the hippocampal slice, and supports studies showing the efficacy of dextromethorphan in animal models of ischemia [6]. This protective effect is similar to that reported t\~r AP-5. although relatlx.e potency would require rigorous dose response comparisons. Incomplete protection was evident in many AP-5 or dextromethorphan experiments in which the PS amplitude did not fully recover, indicating that functional damage still occurred in these treated slices. This observation is consistent with the effects of another N M D A antagonist, ketamine, which only partially reduces ischemic changes m carotid arter> ligated rabbits [9]. A recent report shows that dextromethorphan has effects on cerebral blood flow durmg focal ischemia, which may contribute to its neuroprotectlve effects in vivo [10]. The results obtained with dextromethorphan in the hippocampal slice preparation implicate additional neuroprotective mechanism independent of cerebral blood flow. The combmed effects of dextromethorphan on cerebral blood flow and N M D A blockade may produce greater eflicacy in vivo than in vitro. Lowered temperature produced greater protection from hypoxia than dextromethorphan or AP-5. Even moderate lowering of ACSF temperature (34- 35°C) had equal or greater protectixe effects compared with antagonist treatment (data not ,shown). Excesses of other

neurotransmitters, for example dopamine, are also involved in mechanisms of hypoxia-induced neuronal damage [7]. Compounds that block only the actions of one neurotransmitter may not adequately attenuate all the injurious processes associated with hypoxia. Lowered temperature may have blocking effects on more than one transmitter, such as on both glutamate and dopamine. This may explain why' lowered temperature prorides greater protection in comparison to N M D A antagonist,s. The protective activit,,, of dextromethorphan has prompted interest in this compound lk~r use climcally as a cerebroprotectant, possibly' in stroke victims. The present work suggests a role for dextromethorphan in preserving neuronal function. This investigation also demonstrates that the hippocampal slice preparation prorides useful reformation about hypoxic damage and its possible mediation by' N M D A receptors. I Ben\emote, H., Drejer, J , Schousboe, A and Dtemer, N H , Lle,,alain of the extracellular concentratlon~ of glutamale and aspartate m rat hJppocampu~ dtll'lrlg tlan,,,lelll cerebral lschemla momtored by' mtracerebral mlcrodlalysl.~. J Ncurochcm , 43 I 1987) 1369 1374. 2 Chol. D W', Maulacc>Gcdde, M A and Krlegstem, A R , Glulamarc ncurotoxlclt) m comcal cell ~.ullure, .l Neuroscl, 7 (1987) 357 36S 3 ( ho~. D W. Dexlrorphan and dcxlromclhorphan attenuate glulamate ncuroto\lclt 5. Brain Res, 403 (19,S7) 333 33(~ 4 Clark. ( i D and Rothman, S M , Blockade of CXCltatory amino acid receptor.,, prolecls ano\lc hlppocampal shoes, Neurosclent_c, 21 (19871665 671 5 ('otman, (' W, Flatman, J A , (}anoilg, A H and Perkins, M.N , Effect,, of e\cltdtor} amino acid dnlagolll~,D, on t2~,oked and spontdncou~ excitatory potenlmls in gUlllea-plg hlppocainpu~, .1. Ph),qol , 37S 11986)403 415 6 George, C P. Goldberg, M P, Chol, D W and Steinberg, ( , . K , Dextromethorphan redtlces ncocorllcal lschemlc neuronal d~tnlage m xlxo, Brain Res., 440 (1987) 375 379 7 Globu.s, M H T , Buqo, R , Dalton l)lctiich, \~' \'aides, E M I and Gmsbelg, M . D , Intra-lschemlc exlracellular relcasc of dopa1-11111c and glutamate IS a,,soclated \~tttl qrtatal \ ulncrablhtv m ischore,a, Ncuroscl Lctt, 91 l lgSS) 36 4() 8 Goldbmg, M P, Pham, PC and ('ho~. D W , Dextrorphan and dcxttomcthorphan block anoxlc mlur_x in cullure, Neurolog), 37 (lq87) 251) 9 Marcou\, F W . Goodrich, J E and Domlmck, M , \ . Kelammc pre,,ents 1,,chem,c neuronal mjur,,, Brain Re~, 452 (1988) t29 335 I() Steinberg, G K., Lo, E H , Kunl~,, D M and Grant. G I)extromeIhorphan alters cerebral blood flow and protecls agamsl cerebral m.lur) after local cerebral l,;chcmla, Soc Neurowl Abstr. 16 (19911) 1278 11 T a n l m o l o , M and Okada, 5', "I'hc ploleclt\c effect', of hypothermla on h~ppocampal shoes from guinea p~g during deprivation of oxygen and glucose. Brain Res, 417 119s7) 239 246 12 Wong. B "Y, Coulter, D A , Cho~. D ',~' and Prince, D A l)extrorphan and dextromethorphan, common ant,tuss~\es, are anl,eptlepla, and antagomTe \;-meth)l-D-asparlatc m brain shoes. Neuroscl l.en,85(1988)261 266