Materials and Methods

Animals—Experiments were conducted on 7 gelding Shetland ponies. The ponies were 4 years old with a mean ± SD body weight of 121 ± 25 kg. Ponies were judged to be healthy on the basis of results of physical, biochemical, and hematologic examinations. Food was withheld from the ponies for 24 hours before the experiments, but they had ad libitum access to water. Two years before the study reported here was conducted, the left carotid artery of each pony was surgically translocated to a subcutaneous position. The Committee for Animal Experimentation, County of Berne, Switzerland, approved the study, which was part of a larger investigation on determination of isoflurane MAC in ponies.

Induction and monitoring of anesthesia—Oxygen was administered to each pony. Anesthesia was then induced by administration of isoflurane^a in oxygen via a face mask by use of a conventional circle anesthetic system.^b The isoflurane vaporizer setting was 1% for 1 minute and then increased to 3% until endotracheal intubation could be performed. For the first 30 minutes, the end-tidal concentration of isoflurane was set at approximately 1.3%, which is the established MAC for equids,¹³ to permit instrumentation. Mechanical ventilation (ie, intermittent positive-pressure ventilation) was started, with initial settings of 8 breaths/min and a tidal volume of 12 mL/kg.

Catheters were inserted into a saphenous vein and carotid artery; these catheters were used for administration of lactated Ringer's solution and monitoring of arterial blood pressure. Esophageal body temperature, ECG, pulse oximetry, arterial blood pressure, inspired and end-tidal oxygen concentration, end-tidal carbon dioxide concentration, and end-tidal anesthetic concentration were continuously monitored by use of a calibrated unit.^c Mean arterial pressure was maintained at > 70 mm Hg by IV administration of dobutamine.^d Arterial blood gas analysis was performed after insertion of the catheter into the carotid artery, and the analysis was then repeated at intervals of 1 hour.

MAC measurement—After instrumentation, the endtidal isoflurane concentration was set at 0.9% and maintained constant for at least 30 minutes. Thereafter, the iMAC for each pony was determined by applying a supramaximal electrical stimulation^e (90 V and 5 Hz) on the oral mucous membranes, as described elsewhere.¹³ Stimulation was applied for 60 seconds 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 anesthetic concentration was increased or decreased by 0.1% end-tidal concentration. After an equilibration period of 30 minutes, electrical stimulation was again applied. This process was continued until anesthetic concentrations were detected that barely permitted and barely prevented, respectively, purposeful movement. The mean of these concentrations constituted a crossover point. The final iMAC was the mean of 3 crossover points. The mean of the iMAC values was designated as the group MAC.

Electrophysiologic recordings and quantification of EMG reflex activity—After each pony was anesthetized and positioned in right lateral recumbency, 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^f and EMG recordings.^g Resistance of stimulation electrodes had to be < 2 K[.Omega]. The ground electrode was placed on the dorsum of each pony at a location immediately caudal to the point of the shoulders. Stimulations and recordings were performed by use of a computerized system, as described elsewhere.¹² The EMG signals were amplified with an overall gain of 5,000 and band-pass filtered (7 to 200 Hz; first-order active filters with a slope of 6 dB/octave). Signals were subsequently passed through an analogue-to-digital converter and stored on a computer for additional processing.

Each stimulus consisted of train-of-five, 1-millisecond, constant-current, square-wave pulses delivered at a frequency of 200 Hz. This configuration has been used for studies^11,14–17 conducted in conscious humans and horses. The final stage of the stimulation was provided by a battery-powered, optoisolated, constant-current stimulator with a maximum supply voltage of 100 V.

For each pony, 3 series of stimulations were performed for isoflurane administered at concentrations of approximately 1.0 MAC (2 concentrations less than the isoflurane MAC [0.85 and 0.95 iMAC] and 1 concentration greater than the isoflurane MAC [1.05 iMAC] in end-tidal concentration increments of 0.1%), always after an equilibration period of at least 30 minutes. 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 by visual observation, and responses were recorded.

To record and quantify EMG reflex activity in response to stimulation, the EMG recording was conducted from 100 milliseconds before until 400 milliseconds after a stimulus, which resulted in a total recording time of 500 milliseconds with 512 sample points (sampling frequency, 1,024 Hz). To be considered a reflex response, the EMG burst following stimulation had to be at least 3 times the amplitude of the background activity with a duration of at least 10 milliseconds within the period from 20 to 70 milliseconds after stimulation onset.

At each MAC, the lowest stimulation intensity able to evoke 2 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. Additional electrical stimulations at 3, 5, 10, 20, 30, and 40 mA were delivered in ascending order at 1-minute intervals to assess the intensity-response function. Finally, 60 consecutive stimuli were administered at a fixed intensity of 40 mA and frequency of 1 Hz. The EMG responses obtained were averaged and quantified.

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 reflex (deflection from baseline). To quantify the muscular response, the RMS value for reflex amplitude was calculated for the period from 20 to 70 milliseconds after stimulation. The background EMG amplitude was calculated as the RMS amplitude during the 100-millisecond interval before stimulation. To minimize the influence of possible variability among ponies, the relative amplitude of the reflex in the period from 20 to 70 milliseconds after stimulation was calculated as the ratio between the RMS amplitude detected during such periods and the RMS of the background EMG in the 100-millisecond period before stimulation. During recordings performed as part of preliminary experiments, it was observed that a burst of EMG reflex activity in response to electrical nerve stimulation could appear within the period from 20 to 70 milliseconds after stimulation in ponies at a light plane of isoflurane-induced anesthesia.

Recovery and reflex recordings in standing ponies— After completion of MAC determination and administration of the series of electrical stimulations, ponies were allowed to breath pure oxygen until the return of the swallowing reflex was detected. They were then assisted for recovery. Stimulation and recording electrodes applied during anesthesia were left in place throughout the recovery period. One hour after disconnection from the anesthetic circuit, with the ponies in standing position and residual ataxia resolved, the reflex threshold was again assessed. Intensity of electrical stimulation was initially set at 1 mA and gradually increased in increments of 0.5 mA until a reflex response was detected during the period from 20 to 70 milliseconds after stimulation, as described previously.

Statistical analysis—Nonparametric analysis of data was chosen on the basis of tests for normal distribution. Group MAC was expressed as mean ± SD. Other results were reported as median and IQR (25% to 75%) values.

Relative amplitudes, latencies, and durations of the reflexes at various stimulus intensities and MAC concentrations were analyzed by use of Friedman repeated-measures ANOVA on ranks, with post hoc Tukey tests for multiple comparisons. Values obtained for the 2 muscles were compared by use of the Mann-Whitney rank sum test. Values of P < 0.05 were considered significant. Statistical analyses were performed by use of commercially available software.^h

Results

Anesthetic period—Mean ± SD isoflurane MAC was 1.0 ± 0.2%, with values of iMACs < 1.2% (corrected for a barometric pressure of 760 mm Hg). Mean total duration of anesthesia was 471 ± 37 minutes. Normocapnia and normotension were maintained throughout anesthesia in all ponies, with mean endtidal carbon dioxide concentration of 33.9 ± 1.6 mm Hg and mean arterial blood pressure of 92.5 ± 6.8 mm Hg. Mean PaCO² was 37.2 ± 1.5 mm Hg. All ponies recovered uneventfully from anesthesia.

Reflex responses at threshold stimulation intensities—Reflex threshold intensities after anesthesia were always significantly (P < 0.001) less than threshold intensities during anesthesia for both muscles (Figure 1). Median reflex thresholds in awake ponies were 3 mA (IQR, 3 to 4 mA) for the common digital extensor muscle and 4.5 mA (IQR, 4.0 to 6.5 mA) for the deltoid muscle. During isoflurane-induced anesthesia, 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 manner (Table 1). Median maximal values of 8 mA (IQR, 7.2 to 9.5 mA) for the common digital extensor muscle and 24 mA (IQR, 19 to 41 mA) for the deltoid muscle were found at the highest isoflurane concentration tested (1.05 iMAC). Significantly higher reflex threshold intensities were found for the deltoid muscle, compared with threshold intensities for the common digital extensor muscle, at all tested isoflurane concentrations. However, no significant differences were found for threshold intensities between the 2 muscles after anesthesia in awake ponies. During anesthesia, stimulations at intensities that would barely evoke an EMG reflex response from the common digital extensor muscle were not accompanied by any visible movement of the stimulated limb, whereas stimulations that evoked reflexes from the deltoid muscle were always accompanied by a visible flexion-protraction movement of the stimulated limb. In 2 ponies at 1.05 iMAC, the maximal stimulation intensity of 44 mA was not sufficient to evoke a reflex response and the reflex threshold intensities could not be determined.

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Median and IQR NWR thresholds for the common digital extensor muscle (black circles) and deltoid muscle (white circles) of 7 ponies during anesthesia at 0.85, 0.95, and 1.05 iMAC of isoflurane and in standing ponies after recovery from anesthesia (awake).

Citation: American Journal of Veterinary Research 67, 5; 10.2460/ajvr.67.5.762

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At threshold intensities, relative amplitudes of the reflex responses were similar for both muscles during and after anesthesia and no concentration-related changes were observed (Figure 2). Latency of the reflex responses remained stable and did not change significantly (P = 0.600) for the common digital extensor muscle, with a median overall value of 20 milliseconds (IQR, 19 to 22 milliseconds). Conversely, latency of the reflex response for the deltoid muscle had significant changes, decreasing from a median value of 33 milliseconds (IQR, 31 to 36 milliseconds) during anesthesia to 19 milliseconds (IQR, 16 to 32 milliseconds) after anesthesia. In awake ponies, no significant difference was found for latency of the reflex response between the 2 muscles. Duration of the reflex response did not change during and after anesthesia for the common digital extensor muscle, with a median overall value of 23 milliseconds (IQR, 20 to 25 milliseconds). Conversely, duration of the reflex response for the deltoid muscle increased significantly from a median value of 25 milliseconds (IQR, 22 to 29 milliseconds) during anesthesia to a median value of 38 milliseconds (IQR, 30 to 45 milliseconds) in awake ponies (Table 1).

Reflex responses to stimulations of increasing intensity—During anesthesia and at each MAC tested, amplitudes of the reflex responses from both muscles increased significantly (P < 0.001) as stimulus intensity increased from 3 to 40 mA (Figure 3). Reflex responses were consistently recorded for the common digital extensor muscle but not for the deltoid muscle when stimulations of 5 mA were applied (Table 2). The reflex responses usually consisted of a burst of EMG activity during the period from 20 to 70 milliseconds after stimulation. Graded electrical-evoked reflexes during isoflurane-induced anesthesia were detected at 0.85, 0.95, and 1.05 iMAC (Figure 4). Between 0.85 and 1.05 iMAC, there was concentration-dependent depression of electrically evoked reflexes with a reduction in the slopes of stimulus-response functions. For the common digital extensor muscle, amplitudes of the reflex response decreased significantly for increasing anesthetic concentrations when stimuli were administered at 30 mA. Similarly, a concentration-dependent decrease was observed for stimulations at 20, 30, and 40 mA for the deltoid muscle. Significant differences were always detected between the lowest and the highest isoflurane concentrations.

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Representative NWR threshold recordings obtained for the deltoid muscle of a pony during anesthesia at 0.85, 0.95, and 1.05 iMAC of isoflurane and after anesthesia (awake). Total recording time was 500 milliseconds. The stimulus onset is indicated (arrow).

Citation: American Journal of Veterinary Research 67, 5; 10.2460/ajvr.67.5.762

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Median and IQR relative amplitudes of EMG activity for the common digital extensor muscle (A) and deltoid muscle (B) of 7 ponies calculated for the period 20 to 70 milliseconds after stimulus with 3, 5, 10, 20, 30, and 40 mA during anesthesia induced by administration of isoflurane concentrations of 0.85 iMAC (circles and solid line), 0.95 iMAC (squares and dashed line), and 1.05 iMAC (inverted triangles and dotted line). Relative amplitude is the ratio between the RMS amplitude of the EMG activity in the period 20 to 70 milliseconds after stimulus and the RMS amplitude of the EMG activity in the 100-millisecond interval before stimulus. Notice that the scale of the y-axis differs between panels. The reflex threshold (thin horizontal dashed line) is indicated for each muscle.

Citation: American Journal of Veterinary Research 67, 5; 10.2460/ajvr.67.5.762

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Representative EMG recordings obtained from the deltoid muscle of a pony during anesthesia at isoflurane concentrations of 0.85, 0.95, and 1.05 iMAC after stimulus with 3, 5, 10, 20, 30, and 40 mA. The onset of stimulus is indicated for each iMAC value (vertical dotted lines).

Citation: American Journal of Veterinary Research 67, 5; 10.2460/ajvr.67.5.762

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In general, latencies of the reflex responses during isoflurane-induced anesthesia decreased with increasing stimulation intensities (Table 2). Significance of this pattern was detected at each isoflurane concentration for the reflex response of the common digital extensor muscle. Duration of the reflex responses tended to increase with increasing stimulation intensities, with significant differences between responses for the common digital extensor muscle at 0.85 and 0.95 iMAC. At 1.05 iMAC, duration of the reflex responses remained stable regardless of the stimulation intensity.

Averaged reflex responses—The averaged reflex responses to 60 stimuli administered at 40 mA and 1 Hz during isoflurane-induced anesthesia permitted visual interpretation of 2 reflex components with differing latencies. Median latencies of the 2 reflex components were 19 milliseconds (IQR, 17 to 20 milliseconds) and 39 milliseconds (IQR, 39 to 40 milliseconds) for the common digital extensor muscle and 19 milliseconds (IQR, 17 to 21 milliseconds) and 36 milliseconds (IQR, 36 to 40 millisecond) for the deltoid muscle. Averaged reflex responses appeared to be unaffected by isoflurane concentration because there was no change in latency, amplitude, or duration. Conversely, purposeful movement during the series of 60 stimuli was a concentration-dependent event. At 0.85 iMAC, 4 of 5 ponies had purposeful movement (the other 2 ponies were judged to be at too light of a plane of anesthesia to tolerate stimulation), whereas at 0.95 iMAC, 2 ponies had purposeful movements and 5 did not. None of the ponies had purposeful movement at 1.05 iMAC. In contrast, reflex limb withdrawals in response to stimulation were always evident, beginning with the second or third stimulus.

Discussion

The study reported here revealed that subtle anesthetic-induced changes in sensory-motor processing in ponies anesthetized with isoflurane at concentrations of approximately 1.0 MAC can be detected by assessment of NWR. In fact, in ponies anesthetized at a light plane of anesthesia, NWR thresholds increased with increasing isoflurane concentrations. Furthermore, reflex activities in response to single electrical stimuli were largely depressed at isoflurane concentrations able to prevent purposeful movements in response to supramaximal noxious stimulations, which indicated parallel inhibition of the 2 evoked motor patterns (flexion withdrawal and complex limb movements) in ponies.

Although isoflurane abolishes the electrically induced NWR in humans at concentrations substantially < 1.0 MAC,⁸ it did not do so in our ponies, even at approximately MAC, which suggested species-specific anesthetic modulation of spinal withdrawal reflexes despite homogeneous interspecific MAC values.¹⁸ At all tested MAC multiples, amplitude of the reflex response increased with stimulus intensity, whereas the slope of the stimulus-response function was reduced with increasing isoflurane concentrations. Similarly, concentrations of isoflurane at approximately 1.0 MAC decreased the force of reflexive limb withdrawals in response to noxious thermal stimuli in a concentration-dependent manner in rats, with the largest reduction between 0.9 and 1.1 MAC.⁹ It can be concluded that abolishment of gross purposeful movement is paralleled by inhibition of withdrawal reflexes.

Determination of MAC depends on detection of complex gross purposeful movements, which typically involve the limbs and head. These complex movements are probably dependent on the central pattern generator, which is the neural network involved in processing this behavior.⁶ The central pattern generator receives afferent inputs from the spinal cord and brain and acts to initiate and terminate complex movements.¹⁹ Isoflurane-induced disruption of activity and coordination of the central pattern generator has been described²⁰ in spinal cords isolated from lampreys. Unfortunately, only scarce data are available about interspecies differences in organization and control of the central pattern generator²¹ and its sensitivity to anesthetics.⁶

Withdrawal reflexes in awake animals incorporate flexion of the stimulated limb with extension of the contralateral limb.²² It is not currently known whether this reflex remains intact during inhalant anesthesia.⁶ The study reported here provides evidence of reflex activity in ponies anesthetized with isoflurane concentrations of approximately 1.0 MAC that is consistent with the withdrawal reflex pattern described in conscious horses.¹² When single stimuli of increasing intensity were applied during anesthesia, latency of the NWR decreased but duration of the NWR increased, confirming that the reflex undergoes intensity-dependent modulation.

The concentration-dependent increase of the NWR threshold was a muscle-specific phenomenon, with lowest values found for the common digital extensor muscle (a muscle of the forelimb), compared with values for the deltoid muscle (a muscle of the shoulder). Visible flexion of the stimulated limb accompanied NWRs of the deltoid muscle, whereas limb flexion was mostly lacking when NWRs were recorded for the common digital extensor muscle. The common digital extensor muscle consistently was activated at lower stimulation intensities, compared with values for the deltoid muscle. Reflex activity of the common digital extensor muscle had only a moderate increase in threshold, even for the highest isoflurane concentrations, which indicated a low sensitivity to the depressant effects of the anesthetic. Muscle-specific NWR thresholds were not observed in conscious horses in another study,¹¹ in which other muscles of the forelimb were examined, nor were they observed after recovery from anesthesia in the ponies of the study reported here in which results suggested that isoflurane, lateral recumbency, or both must account for the difference in patterns of reflexive muscle activation during anesthesia.

The reflex response recorded from both forelimb muscles was detected during the period from 20 to 70 milliseconds after stimulus. The early part of the reflex was probably attributable to activation of A[.beta] fibers. With a mean distance of 85 cm between the stimulation electrodes and dorsal point of the shoulders, and considering a peripheral conduction velocity of 75 m/s for ponies,²³ an afferent time of 11 milliseconds was expected. Adding an approximate central delay of 5 milliseconds and neglecting the amount of time needed for the motor component of the reflex,¹⁵ the observed minimal latency of 16 milliseconds appears reasonable. The late part of the reflex, which always terminated before 70 milliseconds after the stimulus, could have reflected activation of A[.Delta] fibers, with a conduction velocity between 15 and 35 m/s, which corresponds to an afferent time between 25 and 55 milliseconds. In the study reported here, it was not possible to separate reflex components originating from A[.beta] fibers from those originating from A[.Delta] fibers when examining a single reflex. When 60 consecutive stimuli at the intensity of 40 mA were applied during 1 minute and the reflex responses were averaged, 2 peaks of differing latencies appeared for both muscles, which confirmed that the 2 expected components exist in ponies (similar to results reported for humans²⁴ and horses^11,12) but probably have more intrasubject variation.

The method applied to determine MAC for each pony in the study reported here is considered the standard for equids and was described for the first time in 1977.¹³ It consists of supramaximal electrical stimulation of the oral mucous membranes; the stimulation is applied at a fixed voltage and frequency during 1 minute or until a gross purposeful movement is observed. Repetition of the stimulus causes a temporal summation of the nociceptive input, which influences MAC values of the volatile agent.²⁵ A similar temporal summation effect was evident when 60 transcutaneous stimuli at 40 mA were applied for 1 minute over the digital nerve and was responsible for the purposeful movements observed during the stimulation series in ponies receiving isoflurane at concentrations < 1.0 MAC. As expected, none of the ponies had purposeful movements when anesthetized at 1.05 iMAC, whereas NWRs still were facilitated as a result of stimulus repetition. The MAC values determined for each of the Shetland ponies of the study were less than the MAC values reported for horses.¹³ Similarly, lower MAC values for ponies, compared with MAC values for horses, have been found for desflurane,²⁶ and large differences in halothane MAC have been reported for horses,^13,27 even when the same standard stimulation method was applied. Among the possible causes for MAC variations, it is likely that variation in sensitivity among subjects, genetic influences,²⁸ failure of the noxious stimulus to generate a supramaximal effect,^29,30 and the point in time that the stimuli were applied in relationship to the time of induction of anesthesia³¹ played a role in these effects.

Quantification of NWR during anesthesia provides information that sensory-motor depression as a whole is evident at various isoflurane concentrations but does not permit differentiation of the effects of anesthesia on afferent and efferent reflex pathways. An invasive approach in laboratory animals has been used to examine the selective effects of inhaled anesthetics on neurons in the dorsal horn³² and motoneurons.¹⁰ Depression of neurons in the dorsal horn of rats is mainly at concentrations of isoflurane < 1.0 MAC.⁹ Analysis of such findings suggests that the immobilizing action of isoflurane is not mediated by depression of nociceptive transmission through the dorsal horn, but is mediated instead by depression at a more ventral location. Isoflurane modulation of the F wave and H reflex as noninvasive measures of motoneuron excitability has been investigated in humans^33,34 and rats.³⁵ These methods have revealed that a prevalent inhibition of the efferent nociceptive pathways is probably responsible for isoflurane-induced immobility evident at concentrations of approximately 1.0 MAC.

In addition to theoretic considerations on the mechanisms of action of isoflurane on the motor system, results of the study reported here provide a practical tool to be applied in veterinary anesthesia for research purposes that will complement studies of MAC. Analysis of our results indicates that a quantitative noninvasive assessment of sensory-motor depression of a simple NWR of a limb at isoflurane concentrations of approximately 1.0 MAC is feasible in ponies. In the future, it will be possible to apply this method to compare the effects of various inhalant agents or anesthetic combinations on spinal processing at concentrations related to MAC in equines and other species, thus improving our knowledge of agent-specific and species-specific mechanisms of anesthetic action.