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The effects of isoflurane minimum alveolar concentration on withdrawal reflex activity evoked by repeated transcutaneous electrical stimulation in ponies
The Veterinary Journal 183 (2010) 337–344
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The effects of isoflurane minimum alveolar concentration on withdrawal reflex activity evoked by repeated transcutaneous electrical stimulation in ponies Claudia Spadavecchia a,*, Olivier Levionnois a, Peter Kronen a, Ole K. Andersen b a b
Anaesthesiology Section, Department of Clinical Veterinary Sciences, Vetsuisse Faculty, Bern University, Bern, Switzerland Centre for Sensory-Motor Interaction, Aalborg University, Aalborg, Denmark
a r t i c l e
i n f o
Article history: Accepted 22 December 2008
Keywords: Isoflurane Withdrawal reflex Electrical stimulus Electromyography Equine
a b s t r a c t The aim of this study was to quantify the effects of isoflurane at approximately the minimum alveolar concentration (peri-MAC) on the temporal summation (TS) of reflex activity in ponies. TS was evoked by repeated electrical stimulations applied at 5 Hz for 2 s on the digital nerve of the left forelimb of seven ponies. Surface electromyographic activity was recorded from the deltoid and common digital extensor muscles. TS thresholds and amplitude of response to stimulations of increasing intensities were assessed during anaesthesia at 0.85, 0.95 and 1.05 times the individual MAC, and after anaesthesia in standing animals. Under isoflurane anaesthesia, TS thresholds increased significantly in a concentration-dependent fashion and at each isoflurane MAC, the responses increased significantly for increasing stimulation intensities. A concentration-dependent depression of evoked reflexes with a reduction in the slopes of the stimulus–response function was observed for both muscles. The results demonstrated that with this model it is possible to describe and quantify the effects of anaesthetics on spinal sensory-motor processing in ponies. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction Attenuation of motor responses induced by anaesthetics is an essential component of the anaesthetic state. Recent evidence has suggested that inhaled anaesthetics, specifically isoflurane, act on the spinal cord to suppress the movement that occurs in response to a noxious stimulus, mainly by inhibiting ventral horn activity (Zhou et al., 1998; Antognini et al., 1999a, 2000, 2003; Antognini and Wang, 1999). The standard method for determining the potency of anaesthetic drugs, the minimal alveolar concentration (MAC), is based on effects on the motor system. The motor response evoked by noxious afferent input during MAC assessment consists of two distinct patterns, namely, a simple flexion withdrawal of the stimulated body part, and complex movements involving multiple body parts, usually described as gross purposeful movements (Quasha et al., 1980). These patterns use differing neural circuits, which possibly undergo differing modulation by volatile anaesthetics (Antognini et al., 1999b). Traditionally, only the suppression of the gross purposeful movement is considered the end-point in MAC determination. The MAC is therefore a non-quantitative all-or-none measure of motor output that does not allow for quantitative detailed analysis * Corresponding author. Tel.: +41 31 631 27 80; fax: +41 31 631 26 20. E-mail address: [email protected] (C. Spadavecchia). 1090-0233/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.12.011
of graded anaesthetic induced changes in sensory-motor processing (Jinks et al., 2003). Despite little research, the systematic evaluation of anaesthetic depressant effects on the simple withdrawal motor pattern following noxious stimulation has been shown to provide interesting information (Jinks et al., 2003). By simultaneously recording the responses of single dorsal horn neurons and hind limb withdrawal force to a graded noxious thermal hind paw stimulation in rats, an anaesthetic-specific site of action was demonstrated: halothane suppressed reflex movement mainly by depressing dorsal horn neurons while isoflurane suppressed movements by an action at more ventral sites in the spinal cord. Whereas the withdrawal reflex of the limb evoked by single electrical stimulation disappeared at concentrations of isoflurane well below the MAC in human volunteers, the facilitation of the reflex following repeated electrical stimulations was still observed at anaesthetic levels around the MAC (Petersen-Felix et al., 1996). This suggested that isoflurane alone is not adequate for inhibiting the central sensitisation that might be evoked by surgical stimuli in humans. Reflex facilitation following repeated stimulation is generally attributed to temporal summation (TS) of the action potentials at spinal level and is classically accompanied by an amplification of subjective pain perception (Arendt-Nielsen et al., 2000). Further experimental evidence indicates that TS plays a basic role in the classical MAC determination process (Dutton et al., 2003), as is demonstrated by an increase in the delay between
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The Committee for Animal Experimentation, County of Berne, Switzerland, approved the study, which was part of a larger investigation on the properties of isoflurane in ponies. Experiments were conducted on seven gelding Shetland ponies. The ponies were 4 years old with a mean ± SD bodyweight (BW) of 121 ± 25 kg. Ponies were judged to be healthy on the basis of physical, biochemical and haematological examinations. Food was withheld for 24 h before the experiments, but water was allowed ad libitum. Two years previously, the left carotid artery of each pony had been surgically translocated to a subcutaneous position. Induction and monitoring of anaesthesia, as well as individual MAC determination have been described in detail elsewhere (Spadavecchia et al., 2006). Briefly, anaesthesia was induced with isoflurane (Isoflo, Abbott AG) in oxygen via a face mask by use of a conventional circle anaesthetic system (Roche electronic respirator 3100, F. Hoffmann-La Roche). Intermittent positive pressure ventilation was performed throughout the experiment. Vital parameters and end-tidal anaesthetic concentrations were continuously monitored using a calibrated unit (S/5 compac, Datex-Ohmeda). Mean arterial pressure was maintained at >70 mm Hg by intravenous (IV) administration of dobutamine (Dobutrex, Eli Lilly). After instrumentation, the end-tidal isoflurane concentration was set at 0.9% and maintained constant for at least 30 min. Thereafter, the MAC for each pony was determined by applying a supra-maximal electrical stimulation (Grass S88, Grass Instruments) at 90 V and 5 Hz on the oral mucous membranes. Stimulation was applied for 60 s or until gross purposeful movement was observed. Lifting of the head or limb movement was interpreted as a positive response, whereas tonic extensions of the limbs or neck were interpreted as a negative response. Depending on the response, the anaesthetic concentration was increased or decreased by 0.1% end-tidal concentration. After an equilibration period of 30 min, electrical stimulation was again applied. This process was continued until two anaesthetic concentrations were detected that barely permitted or prevented purposeful movement. The mean of these concentrations constituted a cross-over point. The final individual MAC (iMAC) was the mean of three cross-over points. The mean of the MAC values for all ponies was designated as the group MAC. The skin over the palmar lateral digital nerve and over the common digital extensor and deltoid muscles of the left forelimb was clipped and cleaned. Pairs of self-adhesive surface electrodes were placed 20 mm apart and used for transcutaneous nerve stimulation (Neuroline 7 00 02-J, Medicotest) and for electromyographic (EMG) recordings (Synapse, Ambu A/S). Resistance of the stimulation electrodes was <2 kX. The ground electrode was placed on the back of each pony at a location immediately caudal to the point of the shoulders. Stimulations and recordings were performed by use of a computerised system, as described previously (Spadavecchia et al., 2004). The final stage of the stimulation was provided by a battery-powered, optoisolated, constant-current device with a maximum voltage of 100 V and a maximal current of 40 mA. The EMG signals were amplified with an overall gain of 5000 and band-pass filtered (7–200 Hz; first-order active filters with 6 dB/octave slope). Signals were subsequently passed through an analogueto-digital converter and stored on a computer for additional processing. Once instrumented for electrophysiological recordings, each pony was stabilised at end-tidal isoflurane concentrations of 0.85, 0.95 and 1.05 times the iMAC. For all ponies the sequence of isoflurane concentrations tested was the same, from the lowest to the highest. At each isoflurane concentration and after at least 30 min equilibration time, two series of electrical stimulations were applied. In all ponies, a series of single stimulations was first performed and a repeated stimulation series was started when the single stimulation series was over. Single stimulations for determination of reflex thresholds and reflex recruitment were firstly delivered and detailed methods and the results of this part of the experiment have been reported (Spadavecchia et al., 2006). Each single stimulus
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Materials and methods
consisted of a train-of-five, 1 ms, constant-current, square-wave pulses delivered at a frequency of 200 Hz. To record and quantify the EMG reflex activity in response to single stimulation, the EMG recording was conducted from 100 ms before until 400 ms after the stimulus, which resulted in a total recording time of 500 ms with 512 sample points (sampling frequency, 1024 Hz). To be considered a reflex response, the EMG burst following stimulation had to be at least three times the amplitude of the background activity with a duration of at least 10 ms within the period 20–70 ms after stimulation onset. Considering a mean afferent distance of approximately 85 cm between the stimulating electrodes and the withers, the early part of the reflex with a latency around 20 ms can probably be attributed to activation of A-beta fibres, with a conduction velocity of 75 m/s (Blythe et al., 1983), while the late part of the reflex, which always terminates <70 ms after the stimulus, could reflect activation of A-delta fibres, with a conduction velocity between 15 and 35 m/ s (Gasser and Erlanger, 1927). At each MAC level, the lowest stimulation intensity able to evoke two consecutive EMG reflex responses was defined as the reflex threshold. Intensity of the current was initially set at 3 mA and increased in increments of 1 mA until a reflex response could be detected for each muscle. When the single stimulation series was over, a repeated stimulation series was started. Repeated electrical stimuli were applied at a frequency of 5 Hz over 2 s (total of 10 stimuli). Each of these stimuli consisted of a train-of-five pulses, previously defined as single stimulus. The intensity of the current was initially set at 3 mA and increased in increments of 1 mA until the TS threshold could be defined (see below). Next, repeated electrical stimulations at 3, 5, 10, 20, 30, and 40 mA were delivered in ascending order at 1 min intervals to assess the stimulus–response function. Reflex EMG responses to stimulations were recorded for the common digital extensor and deltoid muscles of the ipsilateral limb. Evidence of a reflex movement in response to each electrical stimulation was assessed visually. To record and quantify the EMG reflex activity in response to repeated stimulation, EMG activity was stored from 500 ms before until 1500 ms after the stimulation ended, resulting in a total recording time of 4000 ms (sampling frequency, 1 kHz). At each MAC level, the TS threshold was defined as the stimulation intensity able to evoke at least one reflex during the stimulation series as previously defined. To quantify the reflex response, the root-mean-square (RMS) amplitude was calculated for the epochs 20–70 ms after each of the 10 stimuli in the stimulus train. Similarly, the presence of late reflex activity was evaluated by calculating the RMS amplitude for the epochs 70–200 ms. The background EMG amplitude was calculated as the RMS amplitude during the 500 ms interval before stimulation. At each isoflurane concentration and at each stimulus intensity reflexes were assessed by analysing the amplitude of the response to the first stimulus in each train and by calculating the average amplitude of the 10 responses (epochs 20–70 ms after stimulus onset). The absolute TS was calculated by subtracting the RMS amplitude of the first response from the first and each subsequent response and by summing the residuals. Subtracting the initial response from responses to successive stimuli removes the baseline response and leaves only the net response that is facilitated or inhibited during the repetitive stimulus train. The stimulus number evoking the maximal response within the stimulation series was recorded. Latency and duration of reflex responses to the first and the last stimulus of the series of 10 were determined for each pony at the maximal stimulation intensity applied. Latency of the reflex response was defined as the amount of time that elapsed between the onset of the stimulus and onset of the EMG activity burst (deflection from baseline), while duration of the reflex response was defined as the time that elapsed between the onset and offset of the EMG activity burst in the predefined post-stimulation interval. After completion of the stimulations at the three increasing isoflurane concentrations, ponies were allowed to breathe pure oxygen until the swallowing reflex had returned. They were then assisted in recovery. Stimulation and recording electrodes applied during anaesthesia were left in place throughout the recovery period. One hour after disconnection from the anaesthetic circuit, with the ponies in standing position and residual ataxia resolved, single reflexes and TS thresholds were reassessed. The intensity of electrical stimulation was initially set at 1 mA and gradually increased in increments of 0.5 mA until a reflex response could be detected in the interval 20–70 ms after each stimulus, as described previously. A schematic diagram of the course of the events during and after anaesthesia is shown in Fig. 1.
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stimulus onset and motor response observed at inhalant concentrations approaching MAC. We have previously reported that isoflurane at approximately MAC end-tidal concentrations (peri-MAC) depresses withdrawal reflexes evoked by single electrical stimulations in ponies in a concentration-dependent way (Spadavecchia et al., 2006), but does not abolish it completely as is seen in humans (Petersen-Felix et al., 1996). These results suggested that important inter-specific differences in the depressive action of anaesthetics on withdrawal reflexes may exist. The primary aim of the present study was to assess the effects of isoflurane administered at concentrations of approximately one MAC on the TS of reflex activity evoked by repeated transcutaneous electrical stimulations in ponies. A secondary aim was to compare the effects of isoflurane on TS with results previously reported for reflexes evoked by a single electrical stimulation during the same experiment (Spadavecchia et al., 2006).
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Anaesthesia Fig. 1. Schematic diagram of the course of events: repeated stimulations.
Recovery single stimulations;
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C. Spadavecchia et al. / The Veterinary Journal 183 (2010) 337–344 Nonparametric analysis of data was chosen based on tests for normal distribution and the results were reported as median and inter-quartile range (IQR, 25–75%) values. Average amplitude of the responses and the effect of stimulus number on the position of the maximal response were analysed by Friedman repeated-measures ANOVA on ranks, with post hoc Tukey tests for multiple comparisons. Response characteristics measured from the two muscles, threshold intensities for single versus repeated stimulations and first response versus the average response obtained during the stimulation series were compared by Wilcoxon Signed Rank Test. Values of P < 0.05 were considered significant. Statistical analyses were performed using commercially available software (Sigma Stat, version 3.10 for Windows, Systat Software).
Results The mean ± SD isoflurane MAC was 1.0 ± 0.2%, with values of MACs for each pony <1.2% (corrected for barometric pressure of 760 mm Hg). The mean total duration of anaesthesia was 471 ± 37 min. Normocapnia and normotension were maintained throughout anaesthesia in all ponies that recovered uneventfully from anaesthesia. Details of the anaesthesia and MAC determination have been given in our paper reporting the results of the single stimulation series (Spadavecchia et al., 2006). Median single reflex thresholds in awake ponies were 3 mA (IQR, 3–4 mA) for the common digital extensor muscle and 4.5 mA (IQR, 4.0–6.5 mA) for the deltoid muscle. During isoflurane anaesthesia, reflex threshold intensities for both muscles increased significantly (P = 0.007 for the common digital extensor muscle and P = 0.014 for the deltoid muscle) in a concentration-dependent
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manner. Median maximal values of 8 mA (IQR, 7.2–9.5 mA) for the common digital extensor muscle and 24 mA (IQR, 19–41 mA) for the deltoid muscle were found at the highest isoflurane concentration tested (1.05 iMAC) (Fig. 2) (Spadavecchia et al., 2006). TS thresholds in awake ponies were always significantly lower (P < 0.001) than thresholds intensities during anaesthesia for both muscles (Fig. 2). Significantly higher TS thresholds were found for the deltoid muscle, compared to the common digital extensor muscle, at all tested isoflurane concentrations (0.85 iMAC, P = 0.018; 0.95 iMAC, P = 0.026; 1.05 iMAC, P = 0.021) and in awake ponies (P = 0.05). Median TS thresholds in awake ponies were 2.6 mA (IQR, 1.7–2.6 mA) for the common digital extensor muscle and 3 mA (IQR, 1.9–3.4 mA) for the deltoid muscle. During isoflurane anaesthesia, reflex thresholds for both muscles increased significantly (P < 0.001 for the common digital extensor muscle and P = 0.008 for the deltoid muscle) in a concentration-dependent manner. Median maximal values of 7 mA (IQR, 5.1–12.1 mA) for the common digital extensor muscle and 15 mA (IQR, 10.9– 21.3 mA) for the deltoid muscle were found at the highest isoflurane concentration tested (1.05 iMAC). When reflex thresholds to single stimuli and reflex thresholds to repeated stimuli were compared, lower thresholds were found for repeated stimuli compared to single stimuli. Significant differences were detected in awake ponies (P = 0.014) and at 0.85 iMAC (P = 0.003) for the common digital extensor muscle and in awake ponies (P = 0.002), at 0.85 iMAC (P = 0.03) and 1.05 iMAC (P = 0.05) for the deltoid muscle (Fig. 2). Using increasing stimulation intensities up to 40 mA, reflexes could be elicited from both the extensor and the deltoid muscle in all ponies at each isoflurane MAC level. At the maximal stimulation intensity applied, the latency of the reflex response to the first stimulus was significantly longer than the latency of the response to the last stimulus for the muscle deltoid, at the three isoflurane MAC levels (0.85 iMAC, P = 0.016; 0.95 iMAC, P = 0.03; 1.05 iMAC, P = 0.016) and in awake ponies (P = 0.047). Median values of 24 ms (22.5–27 ms) for the first response and 17.5 ms (16– 19 ms) for the last response were found for the pooled data. For the muscle common digital extensor no differences in latency were found between responses to the first and the last stimulus. The observed median latency for pooled data was 19 ms (18–21 ms). For both muscles, no differences in reflex duration were found between responses to the first and the last stimulus. Median values
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Fig. 2. Boxplots showing reflex threshold intensities for the deltoid and common digital extensor (CDE) muscle when single and repeated stimuli were applied to the left digital nerve in seven ponies, anaesthetised with isoflurane at 0.85, 0.95 and 1.05 the individual MAC and after recovery from anaesthesia (awake).*P < 0.05 Lower reflex thresholds for repeated stimuli compared to repeated stimuli.
Fig. 3. Representative recordings from the deltoid muscle of a pony anaesthetised at 0.85 MAC when repeated stimulations (10 stimuli, 5 Hz) were given at 3, 5, 10, 20, 30 and 40 mA. Total recording time was 4 s. Stimulus onset is indicated by an arrow.
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Fig. 4. Representative EMG recordings obtained from the deltoid muscle of a pony during anaesthesia at isoflurane concentrations of 0.85, 0.95 and 1.05 iMAC after stimulation with 3, 5, 10, 20, 30 and 40 mA.
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for pooled data were 38 ms (30–42 ms) and 34 ms (25–40) for the deltoid and the common digital extensor muscles, respectively. During anaesthesia, and at each MAC level, the average reflex amplitude increased significantly with stimulation intensities by 3–40 mA for both muscles (deltoid muscle: 0.85 iMAC P = 0.006; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001; extensor muscle: 0.85 iMAC P = 0.004; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001). Fig. 3 shows an example of a progressive increase in the reflex amplitude when increasing stimulation intensities are given at 0.85 iMAC. Between 0.85 iMAC and 1.05 iMAC, there was a concentration-dependent reflex depression with a reduction in the slopes of the stimulus–response functions for both muscles (Figs. 4 and 5). The highest isoflurane MAC level suppressed the average reflex amplitude compared to the lowest MAC levels in both muscles (P < 0.001). When the average reflex amplitude was compared to the amplitude of the initial response, no significant differences were detected. Absolute TS increased significantly with increasing stimulus intensity only for the common digital extensor at 0.85 iMAC isoflurane (P < 0.001) (Fig. 6). The position of the maximal response within the stimulation series was influenced by the intensity of stimulation, but not by isoflurane concentration for the deltoid muscle (Fig. 6). In the deltoid muscle, the maximal responses occurred after the first stimulus when the stimulation intensities increased (P = 0.033). No clear bursts of late reflex activity were observed under anaesthesia when stimulating at intensities up to 40 mA, rather a diffuse increased EMG activity compared to the pre-stimulation interval was found. The average amplitude of the late reflex activity of both muscles was significantly increased with increasing stimulation intensities at all isoflurane MAC levels (deltoid muscle: 0.85 iMAC P = 0.016; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001; extensor muscle: 0.85 iMAC P = 0.005; 0.95 iMAC P < 0.001; 1.05 iMAC P = 0.002), but there were no differences among isoflurane MAC in responses to stimulations of equal intensities and the extent of increase was much lower than that observed for the epochs 20–70 ms (Fig. 7). The background EMG amplitude remained stable during anaesthesia and no significant differences were found among different isoflurane MAC. Overall, the median amplitude was 4 lV (2– 5 lV) for the deltoid muscle and 3 lV (1–5 lV) for the common digital extensor. The amplitude of the post-stimulation interval (2500–4000 ms) showed a small but statistically significant trend to rise with increasing stimulation intensities (P < 0.001 for both muscles), but this was not significantly related to isoflurane concentration. Complex purposeful movements in response to repeated electrical stimulations were observed in 5/7 ponies only
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Stimulation intensity [mA] Fig. 5. Median and IQR root-mean-square amplitude of averaged reflexes recorded from the deltoid and common digital extensor (CDE) muscle in the 20–70 ms poststimulation intervals. Repeated stimulations were given at 5 Hz over 2 s with intensities of 3–40 mA. # P < 0.05: for a constant isoflurane concentration, the average reflex amplitude increased significantly with increasing stimulation intensities. *P < 0.001: the average reflex amplitude was significantly more suppressed at 1.05 iMAC than at 0.85 iMAC.
at 0.85 iMAC isoflurane. Reflex withdrawal movements always accompanied EMG reflex activity recorded from the deltoid, whereas if reflex activity was recorded from the common digital
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Fig. 6. Median root-mean-square amplitude of reflexes recorded after each stimulus from the deltoid (left column) and from the common digital extensor (CDE, right column) muscles in the 20–70 ms post-stimulation intervals. Ten stimuli were given at 5 Hz over 2 s with intensities of 3–40 mA, at 0.85, 0.95 and 1.05 iMAC. *P < 0.001: absolute temporal summation increased significantly with increasing stimulus intensity.
extensor only, as occurred sporadically, it was not accompanied by visible reflex movements. Discussion The results of the present study indicate that depressive effects of isoflurane on spinal sensory-motor processing can be described and quantified in ponies using a TS model of withdrawal reflexes. In ponies anaesthetised with isoflurane at concentrations of approximately one MAC, TS thresholds increased with increasing isoflurane concentrations. At all tested MAC multiples, the amplitude of the average reflex response increased with stimulus intensity, whereas the slope of the stimulus–response function was reduced with increasing isoflurane concentrations. These results confirmed our previous report for responses to single stimuli during the same experiment,
except that immediately above MAC reflexes to a single stimulation disappeared in some animals as only 5/7 ponies still showed reflexes at 1.05 MAC (Spadavecchia et al., 2006). On the contrary, reflexes to repeated electrical stimulation were not abolished at concentrations that were able to prevent purposeful movements in response to supra-maximal noxious stimulations. This is consistent with the results reported by Petersen-Felix et al. (1995, 1996) who found that TS, but not reflexes to single stimuli, could still be evoked at isoflurane concentrations around MAC in humans. As expected, none of the ponies in our study showed purposeful movements in response to 2 s repeated electrical stimulations when anaesthetised at approximately MAC isoflurane concentration (0.95 and 1.05 iMAC), while spontaneous movements following repeated stimulations were observed below MAC in 5/7 ponies. MAC is the ED50 anaesthetic concentration needed to block gross and purposeful movement evoked by a supra-maximal nox-
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Stimulation intensity [mA] Fig. 7. Median and IQR root-mean-square amplitude of averaged reflexes recorded from the deltoid and the common digital extensor (CDE) muscle in the late reflex epochs (70–200 ms post-stimulation intervals). Repeated stimulations were given at 5 Hz over 2 s with intensities of 3–40 mA. # P < 0.05: at each isoflurane concentration, average reflex amplitude increased significantly with increasing stimulation intensities.
ious stimulus and is considered to be the standard method for determining the immobilising potencies of anaesthetics (Quasha et al., 1980). Traditionally, MAC determination relies on the detection of complex purposeful movements to supra-maximal stimulations involving multiple body parts. These movements are initiated and terminated by the central pattern generator neurons in the spinal cord (Jinks et al., 2005). As facilitation of reflex activity persists at isoflurane concentrations which abolish complex movements, a differential anaesthetic action on the neural networks involved in processing these two movement patterns must be hypothesised. From studies in experimental animals it is known that TS governs part of the MAC of isoflurane and influence anaesthetic requirements (Dutton et al., 2003, 2007). Increasing the duration or frequency of stimulation increases the concentration of isoflurane required to suppress movement by a 0.4 MAC in rats. From an in vitro study it is known that blocking NMDA receptors blocks TS of C-fibre activity, commonly defined as windup (Woolf and Thompson, 1991). Persistence of TS during isoflurane administration might reflect persisting NMDA receptor function (Sonner et al., 2003), although some contrasting evidence has been presented (Yamakura and Harris, 2000). Thus, the immobilising effects of isoflurane seem to be partially independent of its activity on the NMDA receptor (Sonner et al., 2003).
In rats, increasing concentrations of isoflurane from 0.7 to 1.4 MAC uniformly depressed the motor response but had variable effects on neuronal windup recorded from nociceptive lumbar spinal neurons when C-fibre stimulation intensity was used (Jinks et al., 2004). In that study, a generalised reduction in neuronal excitability for increasing isoflurane concentration was hypothesised as the initial C-fibre-evoked responses were reduced in a concentrationdependent manner. In rats, increasing isoflurane from 0.8 to 1.2 MAC significantly depressed the initial C-fibre response but enhanced windup was observed (Cuellar et al., 2005; Ng and Antognini, 2006), again demonstrating that TS effects persist above MAC under isoflurane anaesthesia. As reported for single stimuli, during the 2 s of repeated stimulation, the reflex responses recorded from both forelimb muscles were detected during the epochs 20–70 ms after each stimulus (Spadavecchia et al., 2006), while almost no activity was recorded in the epochs 70–200 ms. Activation of both non-nociceptive and nociceptive fibres seems to account for the reflex recorded during isoflurane anaesthesia in ponies (Spadavecchia et al., 2004, 2006). The stability of the reflex duration observed at different isoflurane concentrations when stimulations at the maximal intensity were given seems to confirm that when the reflex was present both Abeta and A-delta fibres were activated together. The fact that a visible flexion-protraction movement always accompanied reflexes evoked by stimulation intensities above threshold proves that the reflex was at least partly nociceptive in nature. The shortening of the reflex latency observed within the stimulation series for the muscle deltoid may indicate that central facilitation mechanisms are recruited or that the number of the fast conducting fibres recruited increases when stimulations are repeated. No latency shortening was observed for the common digital extensor, which maintained stable reflex characteristics during the stimulation series. We evaluated TS of A fibre activity primarily by calculating the average amplitude of 10 reflex responses obtained during the stimulation series at constant intensity. The average amplitude provides information about the whole effects of repeated stimulation, but does not indicate if there has been an increase or decrease in the amplitude of the reflexes recorded during the stimulation series. For this reason we compared the average amplitude to the amplitude of the response to the first stimulus in each train. The amplitude of the first response should provide information about the baseline excitability of the reflex pathway (Clarke et al., 2002). If the average amplitude was always higher than the amplitude of the first response, it would mean that an increase in the reflex amplitude during the stimulation series consistently occurs. We did not find any significant difference between the average amplitude and the amplitude of the first response, which indicates that in our experiment no consistent increase in reflex amplitude occurred for subsequent stimuli. On the other hand, we looked at the absolute TS as a tool to assess response recruitment characteristics under different conditions. For this parameter, a significant trend to increase was observed for the common digital extensor muscle only at the lowest isoflurane concentration, while at higher concentrations the responses were flattened. This confirms the findings of Dutton et al. (2007) who showed stable but intensity dependent A fibres responses from the sacral dorsal neurons in rats when 20 stimuli up to five times the C-fibre threshold were given. Although reflexes of similar size were obtained during the stimulation series, we observed a difference in the reflex threshold to repeated stimuli compared to the reflex threshold to single stimulus (Spadavecchia et al., 2006). When repeated stimuli were given, a lower stimulation intensity was necessary to evoke at least one reflex during the stimulation series, confirming that a facilitation effect (temporal summation) was actually occurring. In rats anaes-
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thetised with isoflurane up to 1.2 MAC, A fibre responses evoked by repeated electrical stimulations and recorded from dorsal horn neurons remained stable during the stimulation series and were not significantly affected by increasing anaesthetic concentration (Cuellar et al., 2005; Mitsuyo et al., 2006). These latter results are in contrast with our findings, demonstrating a significant depressant effects of A fibre related reflexes when isoflurane was increased from 0.85 to 1.05 MAC. This discrepancy might be due to the fact that we looked at the evoked withdrawal reflex response and not purely at dorsal horn neuronal activity. In general, investigating the modulation of reflexes does not permit differentiation of pharmacological effects on afferent and efferent reflex pathways. An invasive approach in laboratory animals has been used to examine the selective effects of inhaled anaesthetics on neurons in the dorsal horn (Antognini and Carstens, 1999; Jinks et al., 2003) and in the ventral spinal cord (Kim et al., 2007). Depression of dorsal horn neurons by isoflurane occurs mainly well below one MAC (0.4–0.8 MAC) and therefore an isoflurane immobilising action is not attributable to depression of nociceptive transmission through the dorsal horn, but rather by depression of the ventral horn neurons. It has been recently demonstrated that isoflurane, when administered in the periMAC concentration range (0.8–1.2 MAC), depressed motor neurons and pre-motor interneurons to induce immobility in rats (Kim et al., 2007). We found the concentration-dependent increase of TS threshold to be a muscle-specific phenomenon. Consistent with our findings when single stimuli were applied (Spadavecchia et al., 2006), the common digital extensor muscle was activated at lower stimulation intensities than the deltoid muscle, and its activity was less sensitive to the depressant effects of the anaesthetic. Muscle-specific TS thresholds were not seen in conscious horses (Spadavecchia et al., 2004) and after recovery from anaesthesia, which suggests that isoflurane, lateral recumbency, or both must account for the differences in patterns of reflexive muscle activation during anaesthesia. Further studies are needed to clarify this issue. Conclusions The present study has shown that depressive effects of isoflurane on spinal sensory-motor processing in ponies can be described and quantified using the model of TS of withdrawal reflexes. Reflexes following electrical stimulations were still evoked at isoflurane concentrations in the peri-MAC range and a reflex facilitation was observed following the application of repeated stimuli. While a TS paradigm was used as an experimental tool, repetitive noxious stimuli are often applied to equine patients during surgery. So, under isoflurane TS of evoked potentials and windup might still occur. A multimodal approach for intra-operative pain control may decrease the likelihood of developing central sensitisation and postoperative pain in equine patients. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. Acknowledgements The authors would like to thank Dr. Luciano Spadavecchia for developing and manufacturing the electrophysiological equipment and Dr. Marcus Doherr for his assistance in the statistical analysis.
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The effects of isoflurane minimum alveolar concentration on withdrawal reflex activity evoked by repeated transcutaneous electrical stimulation in ponies Claudia Spadavecchia a,*, Olivier Levionnois a, Peter Kronen a, Ole K. Andersen b a b
Anaesthesiology Section, Department of Clinical Veterinary Sciences, Vetsuisse Faculty, Bern University, Bern, Switzerland Centre for Sensory-Motor Interaction, Aalborg University, Aalborg, Denmark
a r t i c l e
i n f o
Article history: Accepted 22 December 2008
Keywords: Isoflurane Withdrawal reflex Electrical stimulus Electromyography Equine
a b s t r a c t The aim of this study was to quantify the effects of isoflurane at approximately the minimum alveolar concentration (peri-MAC) on the temporal summation (TS) of reflex activity in ponies. TS was evoked by repeated electrical stimulations applied at 5 Hz for 2 s on the digital nerve of the left forelimb of seven ponies. Surface electromyographic activity was recorded from the deltoid and common digital extensor muscles. TS thresholds and amplitude of response to stimulations of increasing intensities were assessed during anaesthesia at 0.85, 0.95 and 1.05 times the individual MAC, and after anaesthesia in standing animals. Under isoflurane anaesthesia, TS thresholds increased significantly in a concentration-dependent fashion and at each isoflurane MAC, the responses increased significantly for increasing stimulation intensities. A concentration-dependent depression of evoked reflexes with a reduction in the slopes of the stimulus–response function was observed for both muscles. The results demonstrated that with this model it is possible to describe and quantify the effects of anaesthetics on spinal sensory-motor processing in ponies. Ó 2009 Elsevier Ltd. All rights reserved.
Introduction Attenuation of motor responses induced by anaesthetics is an essential component of the anaesthetic state. Recent evidence has suggested that inhaled anaesthetics, specifically isoflurane, act on the spinal cord to suppress the movement that occurs in response to a noxious stimulus, mainly by inhibiting ventral horn activity (Zhou et al., 1998; Antognini et al., 1999a, 2000, 2003; Antognini and Wang, 1999). The standard method for determining the potency of anaesthetic drugs, the minimal alveolar concentration (MAC), is based on effects on the motor system. The motor response evoked by noxious afferent input during MAC assessment consists of two distinct patterns, namely, a simple flexion withdrawal of the stimulated body part, and complex movements involving multiple body parts, usually described as gross purposeful movements (Quasha et al., 1980). These patterns use differing neural circuits, which possibly undergo differing modulation by volatile anaesthetics (Antognini et al., 1999b). Traditionally, only the suppression of the gross purposeful movement is considered the end-point in MAC determination. The MAC is therefore a non-quantitative all-or-none measure of motor output that does not allow for quantitative detailed analysis * Corresponding author. Tel.: +41 31 631 27 80; fax: +41 31 631 26 20. E-mail address: [email protected] (C. Spadavecchia). 1090-0233/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.12.011
of graded anaesthetic induced changes in sensory-motor processing (Jinks et al., 2003). Despite little research, the systematic evaluation of anaesthetic depressant effects on the simple withdrawal motor pattern following noxious stimulation has been shown to provide interesting information (Jinks et al., 2003). By simultaneously recording the responses of single dorsal horn neurons and hind limb withdrawal force to a graded noxious thermal hind paw stimulation in rats, an anaesthetic-specific site of action was demonstrated: halothane suppressed reflex movement mainly by depressing dorsal horn neurons while isoflurane suppressed movements by an action at more ventral sites in the spinal cord. Whereas the withdrawal reflex of the limb evoked by single electrical stimulation disappeared at concentrations of isoflurane well below the MAC in human volunteers, the facilitation of the reflex following repeated electrical stimulations was still observed at anaesthetic levels around the MAC (Petersen-Felix et al., 1996). This suggested that isoflurane alone is not adequate for inhibiting the central sensitisation that might be evoked by surgical stimuli in humans. Reflex facilitation following repeated stimulation is generally attributed to temporal summation (TS) of the action potentials at spinal level and is classically accompanied by an amplification of subjective pain perception (Arendt-Nielsen et al., 2000). Further experimental evidence indicates that TS plays a basic role in the classical MAC determination process (Dutton et al., 2003), as is demonstrated by an increase in the delay between
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The Committee for Animal Experimentation, County of Berne, Switzerland, approved the study, which was part of a larger investigation on the properties of isoflurane in ponies. Experiments were conducted on seven gelding Shetland ponies. The ponies were 4 years old with a mean ± SD bodyweight (BW) of 121 ± 25 kg. Ponies were judged to be healthy on the basis of physical, biochemical and haematological examinations. Food was withheld for 24 h before the experiments, but water was allowed ad libitum. Two years previously, the left carotid artery of each pony had been surgically translocated to a subcutaneous position. Induction and monitoring of anaesthesia, as well as individual MAC determination have been described in detail elsewhere (Spadavecchia et al., 2006). Briefly, anaesthesia was induced with isoflurane (Isoflo, Abbott AG) in oxygen via a face mask by use of a conventional circle anaesthetic system (Roche electronic respirator 3100, F. Hoffmann-La Roche). Intermittent positive pressure ventilation was performed throughout the experiment. Vital parameters and end-tidal anaesthetic concentrations were continuously monitored using a calibrated unit (S/5 compac, Datex-Ohmeda). Mean arterial pressure was maintained at >70 mm Hg by intravenous (IV) administration of dobutamine (Dobutrex, Eli Lilly). After instrumentation, the end-tidal isoflurane concentration was set at 0.9% and maintained constant for at least 30 min. Thereafter, the MAC for each pony was determined by applying a supra-maximal electrical stimulation (Grass S88, Grass Instruments) at 90 V and 5 Hz on the oral mucous membranes. Stimulation was applied for 60 s or until gross purposeful movement was observed. Lifting of the head or limb movement was interpreted as a positive response, whereas tonic extensions of the limbs or neck were interpreted as a negative response. Depending on the response, the anaesthetic concentration was increased or decreased by 0.1% end-tidal concentration. After an equilibration period of 30 min, electrical stimulation was again applied. This process was continued until two anaesthetic concentrations were detected that barely permitted or prevented purposeful movement. The mean of these concentrations constituted a cross-over point. The final individual MAC (iMAC) was the mean of three cross-over points. The mean of the MAC values for all ponies was designated as the group MAC. The skin over the palmar lateral digital nerve and over the common digital extensor and deltoid muscles of the left forelimb was clipped and cleaned. Pairs of self-adhesive surface electrodes were placed 20 mm apart and used for transcutaneous nerve stimulation (Neuroline 7 00 02-J, Medicotest) and for electromyographic (EMG) recordings (Synapse, Ambu A/S). Resistance of the stimulation electrodes was <2 kX. The ground electrode was placed on the back of each pony at a location immediately caudal to the point of the shoulders. Stimulations and recordings were performed by use of a computerised system, as described previously (Spadavecchia et al., 2004). The final stage of the stimulation was provided by a battery-powered, optoisolated, constant-current device with a maximum voltage of 100 V and a maximal current of 40 mA. The EMG signals were amplified with an overall gain of 5000 and band-pass filtered (7–200 Hz; first-order active filters with 6 dB/octave slope). Signals were subsequently passed through an analogueto-digital converter and stored on a computer for additional processing. Once instrumented for electrophysiological recordings, each pony was stabilised at end-tidal isoflurane concentrations of 0.85, 0.95 and 1.05 times the iMAC. For all ponies the sequence of isoflurane concentrations tested was the same, from the lowest to the highest. At each isoflurane concentration and after at least 30 min equilibration time, two series of electrical stimulations were applied. In all ponies, a series of single stimulations was first performed and a repeated stimulation series was started when the single stimulation series was over. Single stimulations for determination of reflex thresholds and reflex recruitment were firstly delivered and detailed methods and the results of this part of the experiment have been reported (Spadavecchia et al., 2006). Each single stimulus
MAC determination
1.05iMAC
Materials and methods
consisted of a train-of-five, 1 ms, constant-current, square-wave pulses delivered at a frequency of 200 Hz. To record and quantify the EMG reflex activity in response to single stimulation, the EMG recording was conducted from 100 ms before until 400 ms after the stimulus, which resulted in a total recording time of 500 ms with 512 sample points (sampling frequency, 1024 Hz). To be considered a reflex response, the EMG burst following stimulation had to be at least three times the amplitude of the background activity with a duration of at least 10 ms within the period 20–70 ms after stimulation onset. Considering a mean afferent distance of approximately 85 cm between the stimulating electrodes and the withers, the early part of the reflex with a latency around 20 ms can probably be attributed to activation of A-beta fibres, with a conduction velocity of 75 m/s (Blythe et al., 1983), while the late part of the reflex, which always terminates <70 ms after the stimulus, could reflect activation of A-delta fibres, with a conduction velocity between 15 and 35 m/ s (Gasser and Erlanger, 1927). At each MAC level, the lowest stimulation intensity able to evoke two consecutive EMG reflex responses was defined as the reflex threshold. Intensity of the current was initially set at 3 mA and increased in increments of 1 mA until a reflex response could be detected for each muscle. When the single stimulation series was over, a repeated stimulation series was started. Repeated electrical stimuli were applied at a frequency of 5 Hz over 2 s (total of 10 stimuli). Each of these stimuli consisted of a train-of-five pulses, previously defined as single stimulus. The intensity of the current was initially set at 3 mA and increased in increments of 1 mA until the TS threshold could be defined (see below). Next, repeated electrical stimulations at 3, 5, 10, 20, 30, and 40 mA were delivered in ascending order at 1 min intervals to assess the stimulus–response function. Reflex EMG responses to stimulations were recorded for the common digital extensor and deltoid muscles of the ipsilateral limb. Evidence of a reflex movement in response to each electrical stimulation was assessed visually. To record and quantify the EMG reflex activity in response to repeated stimulation, EMG activity was stored from 500 ms before until 1500 ms after the stimulation ended, resulting in a total recording time of 4000 ms (sampling frequency, 1 kHz). At each MAC level, the TS threshold was defined as the stimulation intensity able to evoke at least one reflex during the stimulation series as previously defined. To quantify the reflex response, the root-mean-square (RMS) amplitude was calculated for the epochs 20–70 ms after each of the 10 stimuli in the stimulus train. Similarly, the presence of late reflex activity was evaluated by calculating the RMS amplitude for the epochs 70–200 ms. The background EMG amplitude was calculated as the RMS amplitude during the 500 ms interval before stimulation. At each isoflurane concentration and at each stimulus intensity reflexes were assessed by analysing the amplitude of the response to the first stimulus in each train and by calculating the average amplitude of the 10 responses (epochs 20–70 ms after stimulus onset). The absolute TS was calculated by subtracting the RMS amplitude of the first response from the first and each subsequent response and by summing the residuals. Subtracting the initial response from responses to successive stimuli removes the baseline response and leaves only the net response that is facilitated or inhibited during the repetitive stimulus train. The stimulus number evoking the maximal response within the stimulation series was recorded. Latency and duration of reflex responses to the first and the last stimulus of the series of 10 were determined for each pony at the maximal stimulation intensity applied. Latency of the reflex response was defined as the amount of time that elapsed between the onset of the stimulus and onset of the EMG activity burst (deflection from baseline), while duration of the reflex response was defined as the time that elapsed between the onset and offset of the EMG activity burst in the predefined post-stimulation interval. After completion of the stimulations at the three increasing isoflurane concentrations, ponies were allowed to breathe pure oxygen until the swallowing reflex had returned. They were then assisted in recovery. Stimulation and recording electrodes applied during anaesthesia were left in place throughout the recovery period. One hour after disconnection from the anaesthetic circuit, with the ponies in standing position and residual ataxia resolved, single reflexes and TS thresholds were reassessed. The intensity of electrical stimulation was initially set at 1 mA and gradually increased in increments of 0.5 mA until a reflex response could be detected in the interval 20–70 ms after each stimulus, as described previously. A schematic diagram of the course of the events during and after anaesthesia is shown in Fig. 1.
0.95iMAC
stimulus onset and motor response observed at inhalant concentrations approaching MAC. We have previously reported that isoflurane at approximately MAC end-tidal concentrations (peri-MAC) depresses withdrawal reflexes evoked by single electrical stimulations in ponies in a concentration-dependent way (Spadavecchia et al., 2006), but does not abolish it completely as is seen in humans (Petersen-Felix et al., 1996). These results suggested that important inter-specific differences in the depressive action of anaesthetics on withdrawal reflexes may exist. The primary aim of the present study was to assess the effects of isoflurane administered at concentrations of approximately one MAC on the TS of reflex activity evoked by repeated transcutaneous electrical stimulations in ponies. A secondary aim was to compare the effects of isoflurane on TS with results previously reported for reflexes evoked by a single electrical stimulation during the same experiment (Spadavecchia et al., 2006).
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Reflex recordings
Anaesthesia Fig. 1. Schematic diagram of the course of events: repeated stimulations.
Recovery single stimulations;
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C. Spadavecchia et al. / The Veterinary Journal 183 (2010) 337–344 Nonparametric analysis of data was chosen based on tests for normal distribution and the results were reported as median and inter-quartile range (IQR, 25–75%) values. Average amplitude of the responses and the effect of stimulus number on the position of the maximal response were analysed by Friedman repeated-measures ANOVA on ranks, with post hoc Tukey tests for multiple comparisons. Response characteristics measured from the two muscles, threshold intensities for single versus repeated stimulations and first response versus the average response obtained during the stimulation series were compared by Wilcoxon Signed Rank Test. Values of P < 0.05 were considered significant. Statistical analyses were performed using commercially available software (Sigma Stat, version 3.10 for Windows, Systat Software).
Results The mean ± SD isoflurane MAC was 1.0 ± 0.2%, with values of MACs for each pony <1.2% (corrected for barometric pressure of 760 mm Hg). The mean total duration of anaesthesia was 471 ± 37 min. Normocapnia and normotension were maintained throughout anaesthesia in all ponies that recovered uneventfully from anaesthesia. Details of the anaesthesia and MAC determination have been given in our paper reporting the results of the single stimulation series (Spadavecchia et al., 2006). Median single reflex thresholds in awake ponies were 3 mA (IQR, 3–4 mA) for the common digital extensor muscle and 4.5 mA (IQR, 4.0–6.5 mA) for the deltoid muscle. During isoflurane anaesthesia, reflex threshold intensities for both muscles increased significantly (P = 0.007 for the common digital extensor muscle and P = 0.014 for the deltoid muscle) in a concentration-dependent
25
*
Intensity threshold [mA]
Deltoid 20
15
Repeated stimuli Single stimuus
* 10
*
5
0
manner. Median maximal values of 8 mA (IQR, 7.2–9.5 mA) for the common digital extensor muscle and 24 mA (IQR, 19–41 mA) for the deltoid muscle were found at the highest isoflurane concentration tested (1.05 iMAC) (Fig. 2) (Spadavecchia et al., 2006). TS thresholds in awake ponies were always significantly lower (P < 0.001) than thresholds intensities during anaesthesia for both muscles (Fig. 2). Significantly higher TS thresholds were found for the deltoid muscle, compared to the common digital extensor muscle, at all tested isoflurane concentrations (0.85 iMAC, P = 0.018; 0.95 iMAC, P = 0.026; 1.05 iMAC, P = 0.021) and in awake ponies (P = 0.05). Median TS thresholds in awake ponies were 2.6 mA (IQR, 1.7–2.6 mA) for the common digital extensor muscle and 3 mA (IQR, 1.9–3.4 mA) for the deltoid muscle. During isoflurane anaesthesia, reflex thresholds for both muscles increased significantly (P < 0.001 for the common digital extensor muscle and P = 0.008 for the deltoid muscle) in a concentration-dependent manner. Median maximal values of 7 mA (IQR, 5.1–12.1 mA) for the common digital extensor muscle and 15 mA (IQR, 10.9– 21.3 mA) for the deltoid muscle were found at the highest isoflurane concentration tested (1.05 iMAC). When reflex thresholds to single stimuli and reflex thresholds to repeated stimuli were compared, lower thresholds were found for repeated stimuli compared to single stimuli. Significant differences were detected in awake ponies (P = 0.014) and at 0.85 iMAC (P = 0.003) for the common digital extensor muscle and in awake ponies (P = 0.002), at 0.85 iMAC (P = 0.03) and 1.05 iMAC (P = 0.05) for the deltoid muscle (Fig. 2). Using increasing stimulation intensities up to 40 mA, reflexes could be elicited from both the extensor and the deltoid muscle in all ponies at each isoflurane MAC level. At the maximal stimulation intensity applied, the latency of the reflex response to the first stimulus was significantly longer than the latency of the response to the last stimulus for the muscle deltoid, at the three isoflurane MAC levels (0.85 iMAC, P = 0.016; 0.95 iMAC, P = 0.03; 1.05 iMAC, P = 0.016) and in awake ponies (P = 0.047). Median values of 24 ms (22.5–27 ms) for the first response and 17.5 ms (16– 19 ms) for the last response were found for the pooled data. For the muscle common digital extensor no differences in latency were found between responses to the first and the last stimulus. The observed median latency for pooled data was 19 ms (18–21 ms). For both muscles, no differences in reflex duration were found between responses to the first and the last stimulus. Median values
Awake 0.85 iMAC 0.95 iMAC 1.05 iMAC
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*
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Fig. 2. Boxplots showing reflex threshold intensities for the deltoid and common digital extensor (CDE) muscle when single and repeated stimuli were applied to the left digital nerve in seven ponies, anaesthetised with isoflurane at 0.85, 0.95 and 1.05 the individual MAC and after recovery from anaesthesia (awake).*P < 0.05 Lower reflex thresholds for repeated stimuli compared to repeated stimuli.
Fig. 3. Representative recordings from the deltoid muscle of a pony anaesthetised at 0.85 MAC when repeated stimulations (10 stimuli, 5 Hz) were given at 3, 5, 10, 20, 30 and 40 mA. Total recording time was 4 s. Stimulus onset is indicated by an arrow.
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for pooled data were 38 ms (30–42 ms) and 34 ms (25–40) for the deltoid and the common digital extensor muscles, respectively. During anaesthesia, and at each MAC level, the average reflex amplitude increased significantly with stimulation intensities by 3–40 mA for both muscles (deltoid muscle: 0.85 iMAC P = 0.006; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001; extensor muscle: 0.85 iMAC P = 0.004; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001). Fig. 3 shows an example of a progressive increase in the reflex amplitude when increasing stimulation intensities are given at 0.85 iMAC. Between 0.85 iMAC and 1.05 iMAC, there was a concentration-dependent reflex depression with a reduction in the slopes of the stimulus–response functions for both muscles (Figs. 4 and 5). The highest isoflurane MAC level suppressed the average reflex amplitude compared to the lowest MAC levels in both muscles (P < 0.001). When the average reflex amplitude was compared to the amplitude of the initial response, no significant differences were detected. Absolute TS increased significantly with increasing stimulus intensity only for the common digital extensor at 0.85 iMAC isoflurane (P < 0.001) (Fig. 6). The position of the maximal response within the stimulation series was influenced by the intensity of stimulation, but not by isoflurane concentration for the deltoid muscle (Fig. 6). In the deltoid muscle, the maximal responses occurred after the first stimulus when the stimulation intensities increased (P = 0.033). No clear bursts of late reflex activity were observed under anaesthesia when stimulating at intensities up to 40 mA, rather a diffuse increased EMG activity compared to the pre-stimulation interval was found. The average amplitude of the late reflex activity of both muscles was significantly increased with increasing stimulation intensities at all isoflurane MAC levels (deltoid muscle: 0.85 iMAC P = 0.016; 0.95 iMAC P < 0.001; 1.05 iMAC P < 0.001; extensor muscle: 0.85 iMAC P = 0.005; 0.95 iMAC P < 0.001; 1.05 iMAC P = 0.002), but there were no differences among isoflurane MAC in responses to stimulations of equal intensities and the extent of increase was much lower than that observed for the epochs 20–70 ms (Fig. 7). The background EMG amplitude remained stable during anaesthesia and no significant differences were found among different isoflurane MAC. Overall, the median amplitude was 4 lV (2– 5 lV) for the deltoid muscle and 3 lV (1–5 lV) for the common digital extensor. The amplitude of the post-stimulation interval (2500–4000 ms) showed a small but statistically significant trend to rise with increasing stimulation intensities (P < 0.001 for both muscles), but this was not significantly related to isoflurane concentration. Complex purposeful movements in response to repeated electrical stimulations were observed in 5/7 ponies only
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Stimulation intensity [mA] Fig. 5. Median and IQR root-mean-square amplitude of averaged reflexes recorded from the deltoid and common digital extensor (CDE) muscle in the 20–70 ms poststimulation intervals. Repeated stimulations were given at 5 Hz over 2 s with intensities of 3–40 mA. # P < 0.05: for a constant isoflurane concentration, the average reflex amplitude increased significantly with increasing stimulation intensities. *P < 0.001: the average reflex amplitude was significantly more suppressed at 1.05 iMAC than at 0.85 iMAC.
at 0.85 iMAC isoflurane. Reflex withdrawal movements always accompanied EMG reflex activity recorded from the deltoid, whereas if reflex activity was recorded from the common digital
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Fig. 6. Median root-mean-square amplitude of reflexes recorded after each stimulus from the deltoid (left column) and from the common digital extensor (CDE, right column) muscles in the 20–70 ms post-stimulation intervals. Ten stimuli were given at 5 Hz over 2 s with intensities of 3–40 mA, at 0.85, 0.95 and 1.05 iMAC. *P < 0.001: absolute temporal summation increased significantly with increasing stimulus intensity.
extensor only, as occurred sporadically, it was not accompanied by visible reflex movements. Discussion The results of the present study indicate that depressive effects of isoflurane on spinal sensory-motor processing can be described and quantified in ponies using a TS model of withdrawal reflexes. In ponies anaesthetised with isoflurane at concentrations of approximately one MAC, TS thresholds increased with increasing isoflurane concentrations. At all tested MAC multiples, the amplitude of the average reflex response increased with stimulus intensity, whereas the slope of the stimulus–response function was reduced with increasing isoflurane concentrations. These results confirmed our previous report for responses to single stimuli during the same experiment,
except that immediately above MAC reflexes to a single stimulation disappeared in some animals as only 5/7 ponies still showed reflexes at 1.05 MAC (Spadavecchia et al., 2006). On the contrary, reflexes to repeated electrical stimulation were not abolished at concentrations that were able to prevent purposeful movements in response to supra-maximal noxious stimulations. This is consistent with the results reported by Petersen-Felix et al. (1995, 1996) who found that TS, but not reflexes to single stimuli, could still be evoked at isoflurane concentrations around MAC in humans. As expected, none of the ponies in our study showed purposeful movements in response to 2 s repeated electrical stimulations when anaesthetised at approximately MAC isoflurane concentration (0.95 and 1.05 iMAC), while spontaneous movements following repeated stimulations were observed below MAC in 5/7 ponies. MAC is the ED50 anaesthetic concentration needed to block gross and purposeful movement evoked by a supra-maximal nox-
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Stimulation intensity [mA] Fig. 7. Median and IQR root-mean-square amplitude of averaged reflexes recorded from the deltoid and the common digital extensor (CDE) muscle in the late reflex epochs (70–200 ms post-stimulation intervals). Repeated stimulations were given at 5 Hz over 2 s with intensities of 3–40 mA. # P < 0.05: at each isoflurane concentration, average reflex amplitude increased significantly with increasing stimulation intensities.
ious stimulus and is considered to be the standard method for determining the immobilising potencies of anaesthetics (Quasha et al., 1980). Traditionally, MAC determination relies on the detection of complex purposeful movements to supra-maximal stimulations involving multiple body parts. These movements are initiated and terminated by the central pattern generator neurons in the spinal cord (Jinks et al., 2005). As facilitation of reflex activity persists at isoflurane concentrations which abolish complex movements, a differential anaesthetic action on the neural networks involved in processing these two movement patterns must be hypothesised. From studies in experimental animals it is known that TS governs part of the MAC of isoflurane and influence anaesthetic requirements (Dutton et al., 2003, 2007). Increasing the duration or frequency of stimulation increases the concentration of isoflurane required to suppress movement by a 0.4 MAC in rats. From an in vitro study it is known that blocking NMDA receptors blocks TS of C-fibre activity, commonly defined as windup (Woolf and Thompson, 1991). Persistence of TS during isoflurane administration might reflect persisting NMDA receptor function (Sonner et al., 2003), although some contrasting evidence has been presented (Yamakura and Harris, 2000). Thus, the immobilising effects of isoflurane seem to be partially independent of its activity on the NMDA receptor (Sonner et al., 2003).
In rats, increasing concentrations of isoflurane from 0.7 to 1.4 MAC uniformly depressed the motor response but had variable effects on neuronal windup recorded from nociceptive lumbar spinal neurons when C-fibre stimulation intensity was used (Jinks et al., 2004). In that study, a generalised reduction in neuronal excitability for increasing isoflurane concentration was hypothesised as the initial C-fibre-evoked responses were reduced in a concentrationdependent manner. In rats, increasing isoflurane from 0.8 to 1.2 MAC significantly depressed the initial C-fibre response but enhanced windup was observed (Cuellar et al., 2005; Ng and Antognini, 2006), again demonstrating that TS effects persist above MAC under isoflurane anaesthesia. As reported for single stimuli, during the 2 s of repeated stimulation, the reflex responses recorded from both forelimb muscles were detected during the epochs 20–70 ms after each stimulus (Spadavecchia et al., 2006), while almost no activity was recorded in the epochs 70–200 ms. Activation of both non-nociceptive and nociceptive fibres seems to account for the reflex recorded during isoflurane anaesthesia in ponies (Spadavecchia et al., 2004, 2006). The stability of the reflex duration observed at different isoflurane concentrations when stimulations at the maximal intensity were given seems to confirm that when the reflex was present both Abeta and A-delta fibres were activated together. The fact that a visible flexion-protraction movement always accompanied reflexes evoked by stimulation intensities above threshold proves that the reflex was at least partly nociceptive in nature. The shortening of the reflex latency observed within the stimulation series for the muscle deltoid may indicate that central facilitation mechanisms are recruited or that the number of the fast conducting fibres recruited increases when stimulations are repeated. No latency shortening was observed for the common digital extensor, which maintained stable reflex characteristics during the stimulation series. We evaluated TS of A fibre activity primarily by calculating the average amplitude of 10 reflex responses obtained during the stimulation series at constant intensity. The average amplitude provides information about the whole effects of repeated stimulation, but does not indicate if there has been an increase or decrease in the amplitude of the reflexes recorded during the stimulation series. For this reason we compared the average amplitude to the amplitude of the response to the first stimulus in each train. The amplitude of the first response should provide information about the baseline excitability of the reflex pathway (Clarke et al., 2002). If the average amplitude was always higher than the amplitude of the first response, it would mean that an increase in the reflex amplitude during the stimulation series consistently occurs. We did not find any significant difference between the average amplitude and the amplitude of the first response, which indicates that in our experiment no consistent increase in reflex amplitude occurred for subsequent stimuli. On the other hand, we looked at the absolute TS as a tool to assess response recruitment characteristics under different conditions. For this parameter, a significant trend to increase was observed for the common digital extensor muscle only at the lowest isoflurane concentration, while at higher concentrations the responses were flattened. This confirms the findings of Dutton et al. (2007) who showed stable but intensity dependent A fibres responses from the sacral dorsal neurons in rats when 20 stimuli up to five times the C-fibre threshold were given. Although reflexes of similar size were obtained during the stimulation series, we observed a difference in the reflex threshold to repeated stimuli compared to the reflex threshold to single stimulus (Spadavecchia et al., 2006). When repeated stimuli were given, a lower stimulation intensity was necessary to evoke at least one reflex during the stimulation series, confirming that a facilitation effect (temporal summation) was actually occurring. In rats anaes-
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thetised with isoflurane up to 1.2 MAC, A fibre responses evoked by repeated electrical stimulations and recorded from dorsal horn neurons remained stable during the stimulation series and were not significantly affected by increasing anaesthetic concentration (Cuellar et al., 2005; Mitsuyo et al., 2006). These latter results are in contrast with our findings, demonstrating a significant depressant effects of A fibre related reflexes when isoflurane was increased from 0.85 to 1.05 MAC. This discrepancy might be due to the fact that we looked at the evoked withdrawal reflex response and not purely at dorsal horn neuronal activity. In general, investigating the modulation of reflexes does not permit differentiation of pharmacological effects on afferent and efferent reflex pathways. An invasive approach in laboratory animals has been used to examine the selective effects of inhaled anaesthetics on neurons in the dorsal horn (Antognini and Carstens, 1999; Jinks et al., 2003) and in the ventral spinal cord (Kim et al., 2007). Depression of dorsal horn neurons by isoflurane occurs mainly well below one MAC (0.4–0.8 MAC) and therefore an isoflurane immobilising action is not attributable to depression of nociceptive transmission through the dorsal horn, but rather by depression of the ventral horn neurons. It has been recently demonstrated that isoflurane, when administered in the periMAC concentration range (0.8–1.2 MAC), depressed motor neurons and pre-motor interneurons to induce immobility in rats (Kim et al., 2007). We found the concentration-dependent increase of TS threshold to be a muscle-specific phenomenon. Consistent with our findings when single stimuli were applied (Spadavecchia et al., 2006), the common digital extensor muscle was activated at lower stimulation intensities than the deltoid muscle, and its activity was less sensitive to the depressant effects of the anaesthetic. Muscle-specific TS thresholds were not seen in conscious horses (Spadavecchia et al., 2004) and after recovery from anaesthesia, which suggests that isoflurane, lateral recumbency, or both must account for the differences in patterns of reflexive muscle activation during anaesthesia. Further studies are needed to clarify this issue. Conclusions The present study has shown that depressive effects of isoflurane on spinal sensory-motor processing in ponies can be described and quantified using the model of TS of withdrawal reflexes. Reflexes following electrical stimulations were still evoked at isoflurane concentrations in the peri-MAC range and a reflex facilitation was observed following the application of repeated stimuli. While a TS paradigm was used as an experimental tool, repetitive noxious stimuli are often applied to equine patients during surgery. So, under isoflurane TS of evoked potentials and windup might still occur. A multimodal approach for intra-operative pain control may decrease the likelihood of developing central sensitisation and postoperative pain in equine patients. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. Acknowledgements The authors would like to thank Dr. Luciano Spadavecchia for developing and manufacturing the electrophysiological equipment and Dr. Marcus Doherr for his assistance in the statistical analysis.
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