Characterisation of sevoflurane effects on spinal somato-motor nociceptive and non-nociceptive transmission in neonatal rat spinal cord: an electrophysiological study in vitro

Neuropharmacology 44 (2003) 811–816 www.elsevier.com/locate/neuropharm

Characterisation of sevoflurane effects on spinal somato-motor nociceptive and non-nociceptive transmission in neonatal rat spinal cord: an electrophysiological study in vitro E. Matute a,b, J.A. Lopez-Garcia a,∗ a

Departamento de Fisiologı´a, Universidad de Alcala´, Alcala´ de Henares, Madrid 28871, Spain b Departamento de Anestesia, Hospital Universitario de la Princesa, Madrid 28006, Spain Received 25 June 2002; received in revised form 22 January 2003; accepted 22 January 2003

Abstract Sevoflurane is the latest halogenated ether introduced in clinical anaesthesia, and its effects at the spinal level are not fully characterised. The rat hemisected spinal cord preparation was used to test the effects of sevoflurane on spinal nociceptive and nonnociceptive synaptic transmission as well as on excitations produced by application of glutamate-receptor agonists. Sevoflurane was dissolved in artificial cerebrospinal fluid (ACSF) with a specific vaporiser, and its final concentration was assessed with gas chromatography. Sevoflurane reduced the mono-synaptic reflex (EC50⬇219 µM) and the slow components of the dorsal root–ventral root potentials (EC50⬇72 µM) elicited by single dorsal root stimulation as well as the cumulative depolarisation (CD) elicited by repetitive stimulation (EC50⬇98 µM). AMPA- and NMDA-induced depolarisations were also reduced by sevoflurane (respective EC50s were 206 and 127 µM). Inhibition of NMDA-induced depolarisation was TTX resistant. However, complete blockade of NMDA receptors with d-AP5 did not prevent further reduction of the CD by sevoflurane. All the effects reported were concentration-dependent and reversible. We conclude that sevoflurane applied at clinically relevant concentrations induces a strong depression of nociceptive and nonnociceptive spinal systems, which may be partly mediated by interfering with excitatory amino acid transmission.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Sevoflurane; Halogenated ethers; Spinal cord transmission; Excitatory amino acids

1. Introduction Sevoflurane is a volatile anaesthetic, which belongs to the family of halogenated ethers. Among others, this family includes compounds such as halothane, isoflurane and enflurane, all of which are clinically used as anaesthetics. It is known that all these compounds act at various levels of the central nervous system. Within the last decade the spinal cord has been recognised as an important site of anaesthetic action to reduce mobility in response to surgical noxious stimulation (Rampil et al., 1993; Antognini and Schwartz, 1993). Several

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experimental studies performed in the isolated spinal cord in vitro show that halogenated ethers can depress specific components of ventral root reflexes elicited by dorsal root stimulation (DR-VRRs). Sevoflurane has been shown to depress the mono-synaptic reflex (MSR) (Tsutahara et al., 1996) but no information has been generated regarding the effects of this compound on nociceptive reflexes. The first aim of the present study was to characterise the effects of sevoflurane on synaptic transmission between sensory and motor neurones in a rat spinal cord in vitro preparation. In addition to single dorsal root stimulation, here we use repetitive high intensity stimulation of the dorsal roots, which evokes a C-fibre mediated, frequency-dependent cumulative depolarisation (CD) (Thompson et al., 1992). This is mediated by complex mechanisms and reflects the facilitation of the

0028-3908/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00055-8

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system produced by sustained nociceptive stimulation (Herrero et al., 2000). Halothane, isoflurane and enflurane have been shown to depress N-methyl-d-aspartate (NMDA)- and α-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)receptor mediated responses to different extents and via pre-synaptic or post-synaptic mechanisms (Perouansky et al., 1995; Cheng and Kendig, 2000; Nishikawa and MacIver, 2000). Similarly, these compounds can act on GABA-ergic transmission with different strengths (Nishikawa and MacIver, 2001) and are likely to modulate cellular excitability via actions on different ion channels (MacIver and Kendig, 1991). Therefore it appears that halogenated ethers produce their anaesthetic effects acting at a cluster of related mechanisms and that each compound has its own pattern of action not identical to the others’. The second aim of the present study was to characterise the effects of sevoflurane on NMDA- and AMPAreceptor mediated excitation of motor neurones and to evaluate whether these effects could be involved in the analgesia and reduced mobility produced by this compound during anaesthesia.

2. Materials and methods 2.1. Electrophysiological experiments Neonate (7–10 day old) Wistar rats were anaesthetised with urethane (2 g/kg, i.p.). A dorsal laminectomy was performed to reveal the spinal cord with attached dorsal and ventral roots. The cord was rapidly excised, placed in artificial cerebrospinal fluid (ACSF) and hemisected. The hemisected cord was placed in the recording chamber and continuously superfused with oxygenated (O2/CO2, 95:5%) ACSF. The composition of the ACSF (in mM) was: NaCl 128, KCl 1.9, KH2PO4 1.2, MgSO4 1.3, CaCl2 2.4, NaHCO3 26, and glucose 10 (pH 7.4; recording temperature 23±1 °C). A period of 60 min was allowed for the preparation to stabilise before testing spinal reflexes. The L4 or L5 lumbar dorsal root and the corresponding ventral root were placed in tight-fitting glass suction electrodes. Responses to electrical stimulation of the dorsal root were recorded from the ventral root via a DC coupled amplifier, digitised at 3.3 kHz and stored for off-line computer-aided analysis. Spinal reflexes were elicited with high intensity electrical stimuli (200 µs and 300 µA applied to the dorsal root) previously shown to be about three times the threshold for C-fibre stimulation (Hedo et al., 1999). The stimulation pattern consisted of three single stimuli at 30 s intervals followed by a train of 20 stimuli at 1 Hz. NMDA was bath applied at 50 µM for 45 s and AMPA at 20 µM for 30 s. The compound motoneuronal

response was recorded from the ventral root in the form of a slow depolarisation. Three applications of the same amino acid analogue were made in control ACSF, then a fourth response was obtained in sevoflurane-containing ACSF and one or two more responses were obtained during sevoflurane wash-out. All applications of the same amino acid were performed at 35 min intervals. Some NMDA experiments were performed in the presence of 300 nM tetrodotoxin (TTX) or in the presence of d-AP5 25 µM. All experimental procedures were performed according to European Union and Spanish Government regulations and were supervised and approved by the University Animal Care Facility. 2.2. Administration and quantification of sevoflurane Sevoflurane was vaporised into carbogen at known volume percentages via a specific vaporiser (Quick Fil Dra¨ ger Vapor 19.n). The gas mixture was bubbled into an ACSF-containing reservoir. Sevoflurane-containing ACSF was superfused over the preparation for 30 min periods in order to allow for equilibration. Pilot experiments showed that this period was required to reach maximal effect on responses to synaptic stimulation. The concentration of sevoflurane on the ACSF at the recording chamber was assessed by gas chromatography using a Hewlett-Packard (HP-5890 series II) gas chromatograph and a procedure modified from Lin et al. (1992). For each chromatographic experiment (n=3) a series of standard samples was run followed by a series of bath-collected samples. The data from standard samples were fitted to a linear function using least squares regression and the goodness of fit (r2) was greater than 0.99 in all cases. A linear correlation was found between vaporiser readings and molar concentrations of sevoflurane in ACSF (r2 = 0.995). Concentrations of the bath samples were determined by interpolation in the linear function. These values are clinically relevant and consistent with results reported in vitro by other authors (Park et al., 1996; Nishikawa and MacIver, 2001). 2.3. Measurements and statistical analysis The MSR and slow ventral root potential (VRP) in response to single stimuli were quantified as the mean amplitude from baseline and the mean integrated area with a cut-off at 4 s from stimulus artefact, obtained from three consecutive responses (Hedo et al., 1999). The CD in response to trains of stimuli was incremental. In order to best quantify this response we measured the integrated area under the curve with a cut-off at 24 s from the first stimulus artefact and the rise-rate (calculated as the final amplitude minus the initial amplitude divided by 18 s; Hedo et al., 1999). AMPA- and

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NMDA-induced depolarisations were quantified as the maximum amplitude from baseline. For each variable considered, the difference between the value found during sevoflurane administration and the immediate preceding control was analysed. Differences obtained for a range of sevoflurane concentrations were analysed by means of one-way ANOVA with Dunnett post-hoc tests on raw data. EC50 values were estimated by four parameter curve fitting using an iterative procedure with commercial software (Graph-Pad Prism 3.0, Graph-Pad Software, San Diego, USA). Two-way ANOVA was used to compare pairs of curves (GB-Stat, Dynamic Microsystems Inc.). Data are represented in figures as mean percentage of control values±SEM.

3. Results A total of 51 hemisected cords were used for the experiments. All cords presented spontaneous activity in the form of short-lasting low amplitude depolarisations at low frequency (typical values were 100–120 ms in duration, 0.05–0.07 mV in amplitude and 0.3–0.5 Hz in frequency). Superfusion of sevoflurane-containing ACSF (50–800 µM) decreased or abolished the spontaneous activity of motor neurones (not shown). 3.1. Effects of sevoflurane on spinal synaptic transmission The mean amplitude of the MSR was 4.92±1.63 mV (range 2.4–9.47 mV; n=19). Sevoflurane produced a reversible reduction of the MSR (see Fig. 1A) which was concentration-dependent (overall ANOVA p⬍0.0001; see Fig. 2A). The estimated EC50 was 219 µM (95% confidence limits: 188–254 µM). The reduction of predrug control values was statistically significant for concentrations of sevoflurane ⱖ126 µM (p⬍0.05). The slow VRPs to single stimuli had a mean integrated area of 0.98±0.26 mV s (n=19) and were reversibly and strongly reduced by sevoflurane in a concentration-dependent manner (overall ANOVA p⬍0.0001; see Figs. 1B and 2A). The estimated EC50 for this effect was 72 µM (95% confidence intervals 55–95 µM). Repetitive dorsal root stimulation evoked a CD (see Fig. 1C) with a mean integrated area of 14.90±2.98 mV s (range 6.05–34.58 mV s; n=19) and a mean rise-rate of 10±1 µV/s (range 6–15 µV/s; n=17). Superfusion of sevoflurane-containing ACSF produced a reversible reduction of the integrated area of the CD (see Fig. 1C). This reduction was concentration-dependent (overall ANOVA p⬍0.0001; see Fig. 2A) and the estimated EC50 was 98 µM (95% confidence limits: 71–137 µM). The reduction of pre-drug control values was statistically significant for concentrations of sevoflurane ⱖ46 µM (p⬍0.05). In contrast a significant reduction of the rise-

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rate of the CD was only obtained at concentrations of sevoflurane ⱖ230 µM (up to 30–40% of control values, p⬍0.05, t-test). Sevoflurane applied at 125 µM only reduced the rise-rate to 88±10% of control (not significant, t-test). Two-way ANOVA analysis showed that the effect of sevoflurane on the integrated area of the two nociceptive reflexes studied (single VRPs and CD) was similar. The effect of sevoflurane on nociceptive reflexes was greater than that on the amplitude of the MSR (p⬍0.0001). 3.2. Effects of sevoflurane on NMDA- and AMPAinduced motoneuronal depolarisation Superfusion of NMDA (50 µM) onto the spinal cord for 45 s caused dorsal root depolarisations with a mean amplitude of 0.82±0.16 mV (n=8). Superfusion of sevoflurane-containing ACSF (50–800 µM) produced a significant concentration-dependent reduction of the NMDA-induced depolarisation (overall ANOVA p⬍0.001; see Figs. 2B and 3A) with an EC50 of 127 µM (95% confidence limits: 53–300 µM). Sevoflurane applied at 230 µM reduced the response to NMDA to 36±2% of control amplitude. Responses to NMDA recovered 30 min after sevoflurane wash-out. In order to eliminate indirect effects of NMDA mediated by interneurones we superfused TTX (300 nM) for a period of time sufficient to block spontaneous activity and induced VRPs. After superfusion of TTX the NMDA evoked depolarisation decreased from 0.55±0.05 to 0.26±0.05 mV (n=6). In the presence of TTX, sevoflurane applied at 230 µM produced a reduction of the response to NMDA similar to that found during superfusion with TTX-free ACSF (to 38.90±11.03% of control value; n=4; Figs. 2B and 3B). Superfusion of AMPA (20 µM) directly onto the spinal cord for 30 s evoked a depolarisation lasting typically between 5 and 6 min. The mean peak amplitude of these depolarisations was 0.91±0.30 mV (n=8). Sevoflurane produced a significant decrease of the AMPA-induced depolarisation, which was concentration-dependent (overall ANOVA p⬍0.001; see Figs. 2B and 3C), although the maximal reduction attained was 42±7% of control. The estimated EC50 for sevoflurane-induced depression of responses to AMPA was 206 µM (confidence intervals 110–385 µM). The responses to AMPA returned to normal values after 30 min of wash. A two-way ANOVA showed that responses to NMDA were more strongly reduced by sevoflurane than responses to AMPA (p⬍0.0001). 3.3. Relation between sevoflurane effects on nociceptive transmission and on NMDA-induced depolarisation Although the EC50 values for sevoflurane reduction of the integrated area of the CD and the NMDA-induced

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Fig. 1. Original recordings showing the MSR (A) the slow dorsal root–ventral root reflex (B) and the CD (C) obtained from the same preparation in control ACSF and during superfusion of sevoflurane at different concentrations as indicated. The large vertical lines in the responses to repetitive stimulation correspond to stimulus artefacts. Each trace in A corresponds to the average of three responses. Wash-out of sevoflurane was allowed between applications of sevoflurane but only the final wash is shown for simplicity.

depolarisation are close, a two-way ANOVA performed on both curves detects a significantly greater effect on the CD (p⬍0.0001). In order to further examine a possible relation between these two variables we performed an additional set of experiments using the classical NMDA-receptor antagonist d-AP5. Superfusion of this compound at 25 µM for 30 min produced a complete blockade of NMDA-induced depolarisation and a strong reduction of the rise-rate in responses to repetitive dorsal root stimulation (up to 37±7% of control, t-test p⬍0.05) but only a 49±9% reduction of the integrated area of the CD (n=4). Vaporisation of sevoflurane 230 µM to the d-AP5 containing ACSF produced a significant further reduction of the CD to 24±4% of control (t-test p⬍0.05, n=3). Similarly a comparison between the effects of sevoflurane on the MSR and on the AMPA-induced depolarisation indicate that the former effect is more potent than the later (two-way ANOVA: p⬍0.0001), and this is particularly evident at concentrations of sevoflurane ⱖ230 µM. 4. Discussion The experiments reported here show that sevoflurane inhibits spinal nociceptive and non-nociceptive reflexes at concentrations expected to have anaesthetic effects (Park et al., 1996). The MSR elicited by activation of thick myelinated afferents was less sensitive to sevoflu-

rane than longer latency components of the VRPs which depend on the activation of nociceptive afferents. However, this effect of sevoflurane on the MSR is larger than that reported for isoflurane. It has been shown that isoflurane applied at concentrations that produce a complete blockade of the slow VRP only reduces the amplitude of the MSR by 10% (Savola et al., 1991). In contrast, we have shown that sevoflurane applied at equivalent concentrations (ⱖ230 µM) cause large and significant reductions of the MSR (ⱖ50%). Tsutahara et al. (1996) reported a considerably larger EC50 for this effect of sevoflurane (1.18 mM). Although the discrepancy with our EC50 (219 µM) is large it can only be attributed to differences in experimental conditions such as recording temperature or time of exposure to the anaesthetic. Sevoflurane produced a potent reduction of the CD and the slow VRP to single stimuli, which is comparable to the reductions reported for morphine (Sivilotti et al., 1995) and dexmedetomidine (Kendig et al., 1991). This strong reduction of spinal nociceptive reflexes at anaesthetic concentrations is shared by other halogenated ethers like isoflurane (Savola et al., 1991), whereas propofol has a weaker effect (Jewett et al., 1992). This inhibitory effect of sevoflurane is consistent with the antinociceptive actions of halogenated ethers demonstrated by a decrease of spinal C-fos expression following noxious stimulation (Fukada et al., 1999). In contrast to results reported for isoflurane (Savola et al., 1991), sevoflurane did not show any observable tendency to potentiate nociceptive reflexes at low concentrations,

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Fig. 3. Original recordings show responses to NMDA and to AMPA obtained from different preparations before, during and after superfusion with 230 µM sevoflurane as indicated. Empty and filled squares show the approximate time of application of NMDA and AMPA, respectively. (A) shows a representative example of the effects of sevoflurane on NMDA-induced depolarisation. (B) shows the same sequence during continuous superfusion of TTX-containing ACSF (0.3 µM). (C) shows an example of the effects of sevoflurane on AMPAinduced depolarisation. Fig. 2. The graph in (A) shows quantitative effects of sevoflurane on the MSR, the integrated area of the CD and the integrated area of the dorsal root–ventral root potential (VRP) from pooled data (4–6 observations per data point). The graph in (B) shows quantitative effects of sevoflurane on NMDA- and AMPA-induced depolarisation (3–5 observations per data point). The effect of 230 µM sevoflurane on NMDA-induced depolarisation in the presence of TTX is included for comparison. The concentration of sevoflurane is expressed as log M (lower horizontal axis) and in µM (upper horizontal axis). Overall one-way ANOVA was significant for all variables (see Section 3 for comparisons between curves). Significant differences from respective controls (∗∗ p⬍0.01) and between variables for a given concentration of sevoflurane (## p⬍0.01) are shown as obtained from post-hoc tests.

although this paradoxical effect may require even lower concentrations than those used in the present experiments. Excitatory amino acid receptors are known to mediate DR–VRPs. The MSR is largely dependent on activation of non-NMDA receptors (King et al., 1992). In contrast, the slow potentials originated by activation of high threshold fibres as well as the CD elicited by repetitive stimulation are sensitive to NMDA and neurokinin-receptor antagonists (Thompson et al., 1994). Our results show that sevoflurane depresses NMDA-induced depolarisation in motor neurones in a reversible and concentration-dependent manner as reported for other halogenated ethers (Perouansky et al., 1995; Nishikawa and MacIver, 2000). This depressant effect of sevoflurane was almost identical in the presence of TTX at concentrations sufficient to block synaptic transmission, suggesting a post-synaptic site of action on motor neurones.

We should assume that sevoflurane depresses responses mediated by NMDA receptors located at other spinal interneurones slowing or blocking transmission across poly-synaptic pathways as well as sensory processing. Sevoflurane has been reported to activate a potassium conductance causing hyperpolarisation of hypoglossal motor neurones (Sirois et al., 2000). This mechanism may explain the reduction of responses to NMDA, although the EC50 reported for this effect (290 µM) is larger than that reported here for sevoflurane inhibition of responses to NMDA (127 µM). We have also observed that the effects on NMDAreceptor mediated responses were more sensitive to sevoflurane than those mediated by AMPA receptors. Isoflurane appears to have similar effects (Nishikawa and MacIver, 2000), whereas halothane and enflurane seem to non-selectively depress responses to AMPA and NMDA (Perouansky et al., 1995; Cheng and Kendig, 2000), although conflicting results have been reported in this regard. This observation is in principle consistent with a more potent action of sevoflurane on nociceptive transmission compared to non-nociceptive transmission. Nevertheless, our results indicate clearly that the depressant action of sevoflurane on spinal transmission cannot be explained solely on the basis of modulation of excitatory amino acid-receptor function. Firstly, sevoflurane did not affect the rise-rate of responses to repetitive stimulation as expected from a pure NMDA antagonist (Thompson et al., 1994). Secondly, a complete blockade of NMDA receptors produced only a partial

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reduction of responses to repetitive stimulation, which was further reduced by co-application of sevoflurane. Finally, sevoflurane applied at high concentrations (450– 870 µM) reduces the AMPA-induced depolarisation to about 40% of control, whereas the reduction of the MSR at these concentrations is much larger. From these observations we interpret that reductions caused by sevoflurane on spinal reflexes depend on several mechanisms, which may be recruited at increasing concentrations. A direct or indirect depression of excitatory amino acid-receptor function by sevoflurane is likely to be one of these mechanisms. These spinal effects of sevoflurane may explain the inhibition of movements and the analgesia associated with sevoflurane anaesthesia. Acknowledgements This study was funded by the Spanish Ministry of Science and Technology (SAF-2000-0199), and the Madrid Regional Government (Contrato-Programa) to Prof. F. Cervero´ . The authors wish to thank Dr. J.M.A. Laird and Prof. F. Cervero for comments on the manuscript and Ms. M. Ajubita for technical support. Abbott Laboratories Spain lent a specific vaporiser. References Antognini, J.F., Schwartz, K., 1993. Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 79, 1244–1249. Cheng, G., Kendig, J.J., 2000. Enflurane directly depresses glutamate AMPA and NMDA currents in mouse spinal cord motor neurons independent of actions on GABAA or glycine receptors. Anesthesiology 93, 1075–1084. Fukada, Y., Otsuki, M., Tase, C., 1999. The study of the anesthetic action of halothane on the rat spinal cord by fos immunoreactivity. Masui 48, 966–976. Hedo, G., Laird, J.M., Lopez-Garcia, J.A., 1999. Time-course of spinal sensitization following carrageenan-induced inflammation in the young rat: a comparative electrophysiological and behavioural study in vitro and in vivo. Neuroscience 92, 309–318. Herrero, J.F., Laird, J.M., Lopez-Garcia, J.A., 2000. Wind-up of spinal cord neurones and pain sensation: much ado about something? Prog. Neurobiol. 61, 169–203. Jewett, B.A., Gibbs, L.M., Tarasiuk, A., Kendig, J.J., 1992. Propofol and barbiturate depression of spinal nociceptive neurotransmission. Anesthesiology 77, 1148–1154.

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