Eye (1991) 5, 476-480

Non-Competitive NMDA-Receptor Antagonists and Anoxic Degeneration of the ERG B-Wave In Vitro

JUN C. HUANG, THOMAS E. SALT, MARY J. VOADEN AND JOHN MARSHALL London

Summary Studies have been undertaken to see if the non-competitive NMDA antagonists, keta­ mine, MK-801 and dextromethorphan would preserve the b-wave of the elec­ troretinogram (ERG) in vitro. The drugs had no effect on the ERG b-wave, nor prolonged its survival postmortem. The present results support previous evidence suggesting that NMDA-receptors are not involved directly in synaptic transmission between photoreceptors and ON-bipolar cells. Further, loss of the b-wave in post­ mortem anoxia does not appear to be mediated via NMDA-receptors.

During studies on survival of phototrans­ actions I I in the retina in vitro, and both of duction in postmortem rat and human ret­ these actions can be blocked by non-competi­ inae, we have found that, following tive NMDA-receptor antagonists such as regeneration of visual pigment, PIll ketamine or MK-80l [( + )-5-methyl-l0, 11- responses of the ERG could be recovered to a dihydro-5H -dibenzol a,d]cycIo-hepten-5,10- large extent in previously-bleached, freshly imine]. Therefore, we considered it possible isolated rat retinae, and partially recovered in that loss of the b-wave from the retina post­ human retinae up to 58 hours postmortem.1-3 mortem might also be due to anoxic damage We also noted that the b-wave was apparent involving NMDA receptors. To investigate not only in the rat retina but also in some this, we have attempted to protect rat retinae 2 human retinae up to 43 hours postmortem. .3 against postmortem damage by pretreating Its survival, however, was poor compared rats with systemic injections of ketamine or with PIll. The b-wave is an indicator of syn­ dextromethorphan, and we have also studied aptic transmission between photoreceptors the effects of these drugs and MK-801 on the. and second order retinal neurones, prin­ ERG b-wave in vitro. cipally, if not totally, ON-bipolar cells.�5 Excitatory amino acids mediate the transmis­ Materials and Methods sion and, in man as well as lower order verte­ brates, L-glutamate is likely to play a major role.h-� Recently, it has been reported that Medium dextromethorphan, a non-competitive antag­ Earle's medium (composition in mM-NaCl onist of excitatory amino acid receptors of the 116, KCI 5.4, NaHCO, 26.2, NaH2P04 1.0, NMDA (N-methyl-D-aspartate) type, can MgSO� 0.8, CaCI2 l.8 and glucose 5.5) was protect the rabbit retina against ischaemic pregassed with a moistened mixture of 95% damage and preserve the b-wave in vivo. Y 0/5% CO2 and had a final pH of 7.2 at room Furthermore, NMDA is known to cause temperature. Fetal calf serum was added to a spreading depression!O and to have neurotoxic concentration of 2% for perfusion studies.

Correspondence to: Dr Thomas E Salt, Institute of Ophthalmology, Judd Street, London WCIH 9QS. NMDA ANTAGONISTS AND POSTMORTEM DEGENERATION OF THE ERG B-WAVE 477

The electroretinogram shown to act selectively on ON-bipolar cells Isolated retinae were placed, ganglion cell and thus eliminates the b_wave.17.18 layer downwards, onto Whatman No. 1 filter paper and both surfaces of the tissue/filter Drugs paper preparation were perfused at 7ml/min Ketamine and MK-801 were generous gifts with 400 ml of recirculating perfusion from Parke-Davis Veterinary (Pontypool) medium. The medium was gassed continu­ and Merck, Sharpe and Dohme (Harlow), ously with a moistened mixture of95%, 0/5% respectively. Dextromethorphan and APB were purchased from Sigma Chemicals. CO2 and maintained at 35°C. Photoresponses were recorded with cotton wick and Ag/AgCI Results electrodes, connected to a high input impe­ All experiments were individually performed dance preamplifier with a band pass of with at least three animals. Typical represen­ 0-200 Hz. A 1.5 mm diameter L.E.D. stimu­ tative observations are shown throughout, as lus, wavelength 550 nm, was used. Duration a given treatment protocol resulted in very was 0.2 sec. and maximum intensity (log similar observations in all preparations in a 1= 0) 2.0 X 10-1 quanta/urn". group. Figure I shows that the threshold and Application of ketamine, MK-801 and amplitude of both the a- and b-waves of ret­ dextromethorphan inae from rats which were not pretreated with (1) Pretreatment of rats with ketamine and ketamine were almost unaffected by the pres­ dextromethorphan: ence of up to 300 IlM ketamine in the per­ Female albino Wistar rats, weighing 200 to fusion medium (n = 5). Similarly, MK-80l 300 grams, were maintained in total darkness (up to 200 11M) was without effect (n = 3). overnight before use. Some animals were Figure 2 shows that, at concentrations of 100 injected intraperitoneally with 100 mg/kg and 200 11M, 16 dextromethorphan had no ketamine12 under dim red light, and after effect on the ERG, including the b-wave approximately fiveminutes they had lost their (n = 4). Only when the concentration of dex­ righting and other reflexes. Other animals tromethorphan was increased to 400 or were injected with 40 mg/kg dextromethor­ 800 IlM, did the drug exert a non-specific, phan13 and after 10 minutes the animals, together with the control ones, were anaes­ A Lo,I B C thetised with ether. All three groups were b b 'i�L b --"--- -4.0 -'" � then killed by cervical dislocation, and the 0.25 enucleated eyes stored at room temperature for the times indicated in the text. The Guid­ � -3.' A--- gL � ing Principles in the Care and Use of Animals (DHEW publication, NIH 80-23) were fol­ -2' §L lowed throughout. � � � (2) Application of ketamine, MK-80l and _1.2 §L dextromethorphan in vitro: Ketamine (final concentration 150 to V � � 300 IlM),14 MK-801 (100 to 200 IlM)I) or dex­ tromethorphan (100 to 800 IlM)16 was added to the perfusion medium during measurement Fig. l. Effect of ketamine on the ERG in vitro. The ERG was recorded in a dark-adapted, isolated rat of the ERG. In these experiments control retina. Ketamine was added to the perfusion medium to responses to light stimuli were recorded a final concentration of 150 [!M. A) Before, B) 4 within 5 minutes of the death of the animal. minutes after, and C) 16 minutes after the application of ketamine. Note: increasing the concentration to 300 [!M did not change the responses (not shown). The sweep Application of APB in vitro duration was 2 s and stimulus duration 0.2 s, as For comparison, the effect of DL-APB (2- indicated by the markers beneath the records. amino-4-phosphonobutyric acid) on the ERG Responses obtained at different stimulus intensities was also studied, as this compound has been (Log 1= -4.8 to -1.2) are shown. b = b-wave. 478 JUN CHUANG ET AL.

b taken from rats previously injected with the drug. were left in the dark at room temper­ ature for 30 and 60 minutes. respectively. and then the retinae were isolated. For compari­ control son. eyes from rats without pretreatment with the drug were kept for the same periods of time. Following 30 minutes storage and sub· sequent perfusion with oxygenated medium, 100 J.dl. a b-wave gradually became manifest. taking about 10 minutes to reach its peak of 10 uV: it

then declined (Figure 4) (n = 3). No b-wave � was seen in eyes which had been stored ex vivo

:EL 200 J.JJA for 60 minutes (n = 3). Prior intraperitoneal 0.2 s administration of ketamine or dextromethor­ phan made no difference to the above obser· vations (Figure 4). �L 400 p.)I. Discussion In this study we have used the b-wave of the erg as an index of synaptic transmission from �L J.JJA 800 photoreceptors to second-order retinal cells. Although it is possible that the b-wave arises remotely from these second-order neurones, it is generally agreed that generation of the ��------b-wave involves second-order neurones and Fig. 2. Effect of dextromethorphan on Ihe ERG in that blockade of neurotransmission between vitro. The ERG responses were recorded in a dark­ photoreceptors and ON-bipolar cells abol­ adapted. isolated rat retina before and 10 minutes after applicalions of dexlromethorphan wil/z increasing ishes the b_wave.4517IX The observation of concenlralions. Note: after the responses were partial recovery of the b-wave in the rat retina suppressed. no recovery was ohserved throughold 2 h after 30 minutes anoxia (Figure 4) is in agree­ of repeJfusion with non-dexlromethorphan medium. ment with WinklerlY who reported that a The sweep duralion was 2 s and stimulus duration 0. 2 s. Slimulus intensity: log 1 = -2.4. b = b-wave. b-wave could be regained by re-perfusion A 8 c toxic effect on the responses-irreversibly

suppressing both the b-wave and PIlI com­ -4.8 ponent (Figure 2). Such high concentrations of dextromethorphan are known to exert non­ specific effects. In In addition to the above observations, intraperitoneal injection of 100 mg/kg ketamine prior to tissue isolation had no direct effect on the a- or b-waves of ret­ inae which had been isolated immediately

(n = 3). In contrast to the effects seen with non­ competitive NMDA antagonists, addition of 10 to 100 �M APB to the perfusion medium Fig. 3. Effect of APB on the b-wave in vitro. The reversibly blocked the b-wave (Figure 3) ERG was recorded in a dark-adapted. isolated rat (n = 3), thus showing that the system was retina. APB (finalconcentration 100 �tM) was added fa responsive to a drug that acts selectively on the petfusion medium. as described in the text. A) ON-bipolar cells.l7·ls Before and B) 2 minutes after the application ofAPB. C) 2 minutes after repeJfusion with non-APB medium. To see if ketamine or dextromethorphan Note: reducing the concentralion of APB to 10flM could prqlong survival of the b-wave during produced similar effects Oil the b-w{/\·e. The sweep postmortem anoxia, dark-adapted eyes, duratioll was 2 s and stimulus duratioll 0.2 s. b = b- wave. NMDA ANTAGONISTS AND POSTMORTEM DEGENERATION OF THE ERG 8-WAVE 479

A B receptor antagonists as discussed above, it is unlikely that their postmortem decline involves NMDA-receptor activation. This is, at first sight, somewhat surprising in view of the previous positive finding relating to the b b protection of the b-wave by dextromethor­ � phan upon transient ischaemia in vivo.9 How- -\/ �L � ever, it is possible that different mechanisms 0.2 s underly in vivo ischaemia and postmortem llMxicallMllgl! llr tn!ltdm.�tromb!thorph!ln h!l�

l=31 l=35 other pharmacological effects on the rabbit � � retina than the proposed NMDA-receptor antagonism. According to Yoon and Mar­ mor's observations in the rabbit,9 dextro­ methorphan prevents the photoreceptor cells, -I!� _____ ---II______particularly the outer segments, from degen­ Fig. 4. In vivo pretreatment with ketamine. Dark­ erative changes caused by 60 minutes' ischae­ adapted eyes were kept in the dark at room temperature mia. This phenomenon is interesting, but the for 30 minutes before measurement of the ERG: A) mechanisms of neuro-protection may not be with and B) without prior intraperitoneal injection of via a blockade of NMDA receptors of photo­ ketamine (see the text for details.). The sweep duration was 2 s and stimulus duration 0.2 s. Stimulus intensity: receptor cells, because NMDA has been log 1= -2.7. t = time in minutes after the start of retin'al shown to be ineffective in inducing membrane perfusion. b = b-wave. potential changes in photoreceptor cells.2o.22 It has been shown in animals with a dual ret­ 2 with oxygenated medium within 60 minutes inal circulation (such as the rat,23 gerbil, 4 cat"s anoxia, with no rec.overy if re-perfusion was and monkey26) that the initial degenerative commenced after this 60 minute period. changes, caused by ischaemia of the eye, The present results indicate that ketamine. occur in the inner retina. By contrast, in the MK-801 and dextromethorphan have no rabbit27•28 much of whose retina in effect only direct effect on the b-wave in the rat retina has a choroidal blood supply, interruption of (Figure 1, Figure 2). It is unlikely that the lack the circulation causes ultrustructural of effect on the b-wave is due to an inability of abnormalities in photoreceptor outer seg­ these drugs to penetrate the tissue, as APB ments within 30 minutes and in the inner seg­ was effective in our preparation. A more ments and nuclei within two hours. Thus it likely explanation is that the b-wave is not seems that hypoxic damage to the retina, in mediated via NMDA receptors. This is in terms of the sensitivity of geographic location, agreement with Bloomfield and Dowling20 relates to its in vivo circulation conditions. It who found that NMDLA (N-methyl-DL­ has also been suggested that dextromethor­ aspartate) had no clear effect on rabbit ON­ phan may induce an increase in cerebral blood bipolar cells, and with Coleman and Miller2l flow or a decrease in cerebral metabolic requirements.29 Therefore, it is possible that who showed that D-2-amino-5-phosphonova­ the protection of rabbit photoreceptor cells by leric and D-2-amino-7-phosphonoheptanoic dextromethorphan against ischaemia is acids (competitive NMDA antagonists) have related to a reduction of tissue metabolic rate no effect on the ERG in the mudpuppy. Thus rather than a blockade of NMDA receptors. the current observation confirms that there is This requires further investigation by compar­ little or no synaptically activated NMDA­ ing various species and pharmacological receptor component to ON-bipolar cell agents. responses to light in the vertebrate retina. Since neither the b-wave nor PIlI was bet­ We wish to thank Dr A. Leslie Holden for helpful ter preserved postmortem by pre-treatment discussions. l.C.H. was supported by The South Devon and Cornwall Institution for the Blind. pf rats with ketamine or dextromethorphan Further financial support was provided by the (Figure 4), and both responses were not British Retinitis Pigmentosa Society and the affected by in vitro application of NMDA- American Retinitis Pigmentosa Foundation. 480 JUN C. HUANG ET AI..

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