• View in gallery
    Figure 1—

    Photographs of the hind limbs (left) and caudal lumbar area (right) of a pony in a study of epidurally derived EPs for quantification of caudal nociception. Notice locations of distal (A) and proximal (B) stimulating electrodes and site of insertion of the epidural electrode and catheter (between coccygeal vertebrae 1 and 2 [C]).

  • View in gallery
    Figure 2—

    Mean (thick lines) + SEM (thin lines) EP-EMG waveforms in a pony resulting from 3 baseline recordings with stimulus intensity of 4 mA and stimulus frequency of 0.5 Hz. X-axis values indicate latency from distal stimulation. Amplitude = 100 MV. Vertical lines (x to m) indicate boundaries for RDF calculation.

  • View in gallery
    Figure 3—

    Mean (thick lines in A and C) + SEM (thin lines in A and C) stimulus intensity response characteristics in 7 ponies. A—Evoked potential readings. B—Relative RDF of EP P50. C—Electromyogram readings. D—Relative RDF of EMG N62. RDFrel = Relative RDF, normalized to mean of 0.2- and 0.5-mA stimulations. Y-axis in A and C indicates stimulus onset.

  • View in gallery
    Figure 4—

    Mean (thick lines in A and C) + SEM (thin lines in A and C) methadone response characteristics in 7 ponies. A—Evoked potential readings. B—Relative RDF of EP P50. C—Electromyogram readings. D—Relative RDF of N62 RDF after epidural administration of methadone (0.5 mg/kg) and naloxone (0.04 to 0.06 mg/kg) or saline solution. BL = Baseline. t1 = Time points 5 to 20 minutes after administration of methadone or saline solution. t2 = Time points 25 to 40 minutes after administration of methadone or saline solution. t3 = Time points 45 to 60 minutes after administration of methadone or saline solution. t4 = Time points 65 to 80 minutes after administration of methadone or saline solution. At t3, naloxone (methadone group) or saline solution (saline group) was administered. *Significant (P < 0.05) difference between groups. See Figure 3 for remainder of key.

  • View in gallery
    Figure 5—

    Mean (thick lines in A and C) + SEM (thin lines in A and C) propofol-response characteristics in 4 ponies. A—Evoked potential readings. Y-axis indicates stimulus intensity (mA). B—Relative RDF of EP P50. C—Electromyogram readings. Y-axis indicates stimulus intensity (mA). D—Relative RDF of EMG N62 during administration of propofol (0.5 mg/kg loading dose and 0.1 mg/kg/min constant rate infusion). See Figures 3 and 4 for remainder of key.

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Use of epidurally derived evoked potentials for quantification of caudal nociception in ponies

Johannes P. A. M. van LoonDepartment of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.

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Peter J. StienenDepartment of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.

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Arie DoornenbalDepartment of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands.

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Ludo J. HellebrekersDepartments of Equine Sciences and Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, and Rudolf Magnus Institute of Neuroscience, Utrecht University, 3508 TD Utrecht, The Netherlands.

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Abstract

Objective—To determine whether epidurally derived evoked potentials (EPs) can be used to reliably assess nociception and antinociception in ponies.

Animals—7 ponies.

Procedures—EPs and electromyograms (EMGs) from the quadriceps femoris muscles were recorded simultaneously, following electrical stimulation applied to the distal portion of the hind limb. The effect of increasing stimulus intensity, conduction velocities of the stimulated nerves, effect of epidurally applied methadone, and effect of systemically administered propofol were evaluated.

Results—In the EP and EMG waveforms, 2 distinct complexes, the EP N25 and P50 and the EMG P27 and N62, respectively, were identified. On the basis of their latency and calculated conduction velocities, the EP P50 and EMG N62 were considered to be related to nociception (AD-mediated). All complexes increased significantly in amplitude with increasing stimulus intensity and decreased significantly following epidural administration of methadone or systemic administration of propofol.

Conclusions and Clinical Relevance—Although the experimental setup allowed successful discrimination between tactile- and nociceptive-associated responses, the identified EPs, considered to reflect activity in the spinal cord, could not be definitively differentiated from activity in the lumbosacral epaxial musculature. Further research is required to refine measurement techniques to allow for discrimination between these 2 signals. Similar to other species, neurophysiologic variables such as EPs could potentially become a useful additional tool in quantifying nociception in equidae.

Abstract

Objective—To determine whether epidurally derived evoked potentials (EPs) can be used to reliably assess nociception and antinociception in ponies.

Animals—7 ponies.

Procedures—EPs and electromyograms (EMGs) from the quadriceps femoris muscles were recorded simultaneously, following electrical stimulation applied to the distal portion of the hind limb. The effect of increasing stimulus intensity, conduction velocities of the stimulated nerves, effect of epidurally applied methadone, and effect of systemically administered propofol were evaluated.

Results—In the EP and EMG waveforms, 2 distinct complexes, the EP N25 and P50 and the EMG P27 and N62, respectively, were identified. On the basis of their latency and calculated conduction velocities, the EP P50 and EMG N62 were considered to be related to nociception (AD-mediated). All complexes increased significantly in amplitude with increasing stimulus intensity and decreased significantly following epidural administration of methadone or systemic administration of propofol.

Conclusions and Clinical Relevance—Although the experimental setup allowed successful discrimination between tactile- and nociceptive-associated responses, the identified EPs, considered to reflect activity in the spinal cord, could not be definitively differentiated from activity in the lumbosacral epaxial musculature. Further research is required to refine measurement techniques to allow for discrimination between these 2 signals. Similar to other species, neurophysiologic variables such as EPs could potentially become a useful additional tool in quantifying nociception in equidae.

The epidural technique for analgesia of the caudal portion of the body in horses is widely accepted.1–4 Epidural opioid administration provides regional analgesia with minimal behavioral and gastrointestinal adverse effects.5–7 However, options for valid quantitative assessment of antinociception are limited and primarily focused on NWRs.8–10 Nociceptive withdrawal reflexes provide important but rather limited information on nociception because they rely primarily on afferent dorsal horn activation and subsequent ventral horn motor neuron activation. Suppression of the latter only, consequently resulting in the absence of the spinal reflex, could falsely be interpreted as antinociception, when nociceptive dorsal horn activity and subsequent nociceptive brain center activation remain partially intact.11,12 Valid quantitative assessment of caudal epidural analgesia by recording epidurally derived EPs, rather than NWRs, could potentially overcome this problem.

Evoked potentials are epidurally recorded responses time-locked to stimulation of peripheral somatosensory fibers and are, as such, representative of spinal somatosensory processing. Similar to cortical somatosensory EPs,13,14 changes in EP waveform amplitude or latency may be considered to indicate altered spinal somatosensory processing. Therefore, following noxious stimulation, EPs may potentially be used to detect changes in spinal antinociception. In contrast to various other species, such as rabbits,15 rats,16–19 and humans,20–23 EPs have not been characterized in equidae or evaluated for their applicability to potentially improve the quantification of nociception.

The purpose of the study reported here was to determine whether epidurally derived EPs can be used to reliably assess nociception and antinociception in equidae. Our hypothesis was that by determining the conduction velocities of stimulated nerve afferents, the effect of increasing stimulus intensity, and the effect of epidural opioid and systemic hypnotic drug administration, we could validate our equine nociception technique on the basis of epidurally derived EPs.

Materials and Methods

Animals—The study design was approved by the institutional ethical committee for animal experiments. Selection and subsequent inclusion of the 7 pony geldings (mean ± SD age, 11 ± 1.1 years; weight, 172 ± 13.7 kg) was based on acceptance of handling. The ponies were acclimatized to the experimental room prior to the first experiment. During the experiment, a second pony accompanied the experimental pony to avoid social isolation stress responses.

Instrumentation—The site of insertion of the epidural catheter, between the first and second coccygeal vertebrae, was surgically prepared and locally anesthetized (2 mL of lidocaine HCl 2%,a SC). A 16-gauge Tuohy spinal needleb was inserted into the epidural space. For EP recording, an epidural electrode (custom made from an epidural catheterb) was advanced through the Tuohy needle for 22 to 30 cm up to the lumbosacral junction. A second Tuohy needle was placed epidurally to introduce a 19-gauge catheter for 10 cm for drug administration. While the catheter and the electrode were left in situ, the Tuohy needles were removed and the electrode and catheter were covered with sterile gauzes. Two needle electrodes were placed SC at the level of the lumbosacral junction on the back, one in the median plane (ground electrode) and one 4 cm out of the median plane to the right (reference electrode). Additionally, 3 needle electrodes were placed SC at the level of the right quadriceps femoris muscle with an interelectrode distance of 3 cm to measure the quadriceps femoris EMG. For generating EPs and EMGs, 2 stimulus electrodes (cathode proximal) were placed SC on the distal aspect of the right hind limb, halfway between the coronary band and the metatarsophalangeal (fetlock) joint. For proximal electrical stimulation, 2 stimulus electrodes (cathode proximal) were placed SC on the right hind limb just below the talocrural joint. The positions of the distal and proximal stimulating electrodes and the insertion site of the epidural electrode and catheter were recorded (Figure 1).

Figure 1—
Figure 1—

Photographs of the hind limbs (left) and caudal lumbar area (right) of a pony in a study of epidurally derived EPs for quantification of caudal nociception. Notice locations of distal (A) and proximal (B) stimulating electrodes and site of insertion of the epidural electrode and catheter (between coccygeal vertebrae 1 and 2 [C]).

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.813

Generating and recording of EP and EMG—Following stimulation of the distal portion of the hind limb, EPs and EMGs were recorded simultaneously. Stimuli for a single recording consisted of 32 square-wave electrical stimuli of 0.5 milliseconds' duration each, with an intensity of 0.2 to 4 mA in session 1, and 3 to 4 mA in sessions 2, 3, and 4 at a fixed stimulus frequency of 0.5 Hz. Stimuli were generated by a stimulatorc triggered by software developed by one of the authors (AD). The stimuli were delivered to a stimulation isolation unitd and a constant current unite that controlled the stimulus intensity. The EPs were recorded from the epidural electrode with the 2 needle electrodes close to the lumbosacral junction on the back serving as reference and ground, respectively. Electromyogram was recorded from the 3 needle electrodes (active, reference, and ground electrodes) located at the right quadriceps femoris muscle. Signals were fed to separate but identical bioelectric amplifiers,f amplified 5,000 times, and band-pass filtered between 1.59 and 1,000 Hz. A 50-Hz notch filter was used to eliminate electrical net interference. Subsequently, the analog signals were fed to the data acquisition hardware, digitized online at 10,000 Hz,g and entered into the data acquisition software environment, also responsible for stimulus generation. Each EP-EMG recording consisted of 32 averaged, subsequent responses of 500 milliseconds (10 milliseconds before stimulus and 490 milliseconds after stimulus).

Experimental procedures—Four experimental sessions were performed. In session 1, data were recorded in response to distal stimulation with intensities ranging from 0.2 to 4 mA, administered in random order with the same order for all ponies. After collecting the electrophysiologic responses at the different intensities, data were collected in response to more proximal stimulation, just distal to the talocrural joint, by use of the maximum stimulus intensity (3 or 4 mA). In sessions 2, 3, and 4, electrical stimulation was performed by use of the optimal stimulus intensity for each pony (3 or 4 mA), as established in session 1. In all sessions, 3 baseline runs were recorded with 5-minute intervals. Subsequently, in sessions 2 and 3, either methadoneh (0.5 mg/kg in 10 mL of saline [0.9% NaCl] solution) or a corresponding volume of saline solution was administered epidurally and recordings were performed every 5 minutes for a 40-minute period. Next, naloxonei (0.04 to 0.06 mg/kg in 10 mL of saline solution) or a corresponding volume of saline solution was administered epidurally, and recordings were performed every 5 minutes for another 40 minutes. Four ponies received methadone-naloxone in session 2 and saline solution–saline solution in session 3. In the remaining 3 ponies, the order of treatment was reversed. A minimum of 10 days elapsed between each of the sessions. In session 4, 4 ponies were used. After 3 baseline runs, a slowly administered bolus of propofolj (0.5 mg/kg administered in 1 minute) was administered IV, followed by a constant rate infusion of propofol (0.1 mg/kg/min). After 20 minutes of stabilization, 3 recordings were performed with 5-minute intervals. The ponies were physically supported with a sling during this part of the study. At the end of each session, the ponies were deinstrumented and administered antimicrobials (benzylpenicillink [2 × 105 U/kg] and gentamicinl [6.6 mg/kg, IV]) and an NSAID (flunixin megluminem [1 mg/kg, IV]).

Data and statistical analysis—All data are expressed as mean ± SEM. Statistical analysis was performed by use of commercially available software.n Values of P < 0.05 were considered significant. The EPs and EMG recordings were evaluated by use of the RDF.24 The RDF, an expression of the overall shape of the waveform (EP and EMG) in the latency range studied, is obtained by determining the mean of the absolute differences between all pairs of subsequent sampled data points yk in a specified latency range from x to m, as follows:

article image

Decreases in amplitude and increases in latency of the waveform components will decrease the value of the RDF, whereas increases in amplitude and decreases in latency will increase the value of the RDF. When choosing the RDF latency range (x to m) in the group with the strongest signals (4-mA stimulation intensity) or in the baseline stimulations during the other sessions, individual signals can be analyzed by use of this fixed latency range. The x and m boundaries that were used for calculation of RDFs were determined (Figure 2). Therefore, the RDF is a highly objective method to evaluate EP signals without the need for choosing peak amplitudes in individual signals, which is prone to confounding errors, especially when signals are weak (at low stimulus intensities or during methadone-propofol treatment in the present study).

Figure 2—
Figure 2—

Mean (thick lines) + SEM (thin lines) EP-EMG waveforms in a pony resulting from 3 baseline recordings with stimulus intensity of 4 mA and stimulus frequency of 0.5 Hz. X-axis values indicate latency from distal stimulation. Amplitude = 100 MV. Vertical lines (x to m) indicate boundaries for RDF calculation.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.813

To establish the fiber types involved in the responses recorded in this study, we determined the CV by using the latency shifts for the EP N25/P50, calculated by subtracting the latency obtained after proximal stimulation from the latency obtained after distal stimulation. Subsequently, the CV was calculated per individual animal by dividing the distance between the 2 stimulation sites (12 to 18 cm, measured on the distal portion of the hind limb of every pony separately) by the latency shift of both peaks.

Because we were interested in the AD-afferent transmitted EPs that were elicited in the spinal cord, we investigated the 30- to 80-millisecond latency range. The RDFs were calculated for the most consistent and prominent occurring complexes, that is, for the EPs in the latency range of 30 to 70 milliseconds (EP P50) and for the EMG in the latency range of 52 to 75 milliseconds (EMG N62 [Figure 2]). The EP N25 and EMG P27 were, on the basis of CV data, considered to be generated by stimulation of tactile AB-fibers and were not further analyzed.

For statistical analysis, the RDF values of session 1 were normalized to the mean RDF following stimulation with 0.2 and 0.5 mA for each pony and response (EP-EMG) separately. For session 2, 3, and 4, the RDF values were normalized to their value of the 3 baseline stimulations (relative RDF) and subsequently expressed as the mean of 3 baseline stimulations 5 to 20 minutes after methadone administration (t1), 25 to 40 minutes after methadone administration (t2), 45 to 60 minutes after methadone administration (t3), and 65 to 80 minutes after methadone administration (t4). For session 1, a 1-way RM-ANOVA design was performed with factor intensity. For sessions 2 and 3, a 2-way RM-ANOVA design with factors treatment and time was performed. The RM-ANOVA designs were followed by post hoc analysis when appropriate.

Post hoc tests that were used were 1-way RM-ANOVA design with time and, subsequently, paired t tests. Results from the statistical analysis in sessions 2 and 3 were presented until 60 minutes after methadone administration (t3) because in 1 pony, recording was stopped after 60 minutes because of technical problems. For session 4, a paired t test was performed.

Results

EP and EMG component definition—In the EP-waveform, a negative (EP N25) and a positive (EP P50) complex could be determined (Figure 2). The EMG-waveform consisted of 2 distinct complexes, EMG P27 and EMG N62. It was decided to evaluate the EP waveform with the latency of 28 to 70 milliseconds and the EMG waveform with the latency of 52 to 75 milliseconds as 1 complex.

Determination of CV—Mean latency shifts allowed calculation of CV to be within the AA/B domain (> 35 m/s) for EP N25 and EMG P27 (mean ± SEM, 47.89 ± 7.72 m/s and 70.71 ± 13.06 m/s, respectively) and within the AD domain (4 to 35 m/s) for EP P50 and EMG N62 (mean ± SEM, 25.57 ± 3.03 m/s and 31.10 ± 3.41 m/s, respectively). In 2 animals (pony 2 and 5), no EMG N62 and, thus, no CV based on the EMG could be determined after proximal stimulation. In 1 animal (pony 7), no distinct peaks and, thus, no CV could be determined in both the EP and EMG after proximal stimulation.

Stimulus intensity response characteristics—The RDF EP P50 (P < 0.001) and RDF EMG N62 (P = 0.007) increased significantly with increasing stimulus intensity (Figure 3). All ponies had aversive behavior to some extent, including a withdrawal reflex and full-body movements, during the top range of electrical stimulation.

Figure 3—
Figure 3—

Mean (thick lines in A and C) + SEM (thin lines in A and C) stimulus intensity response characteristics in 7 ponies. A—Evoked potential readings. B—Relative RDF of EP P50. C—Electromyogram readings. D—Relative RDF of EMG N62. RDFrel = Relative RDF, normalized to mean of 0.2- and 0.5-mA stimulations. Y-axis in A and C indicates stimulus onset.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.813

Effect of epidural opioid administration—In 6 of 7 ponies, the 4-mA stimulation intensity was used. In 1 pony, this was not tolerated; therefore, 3-mA stimulation was used. Administration of either methadone or saline solution led to slight and transient (seconds) behavioral responses during the injection phase, characterized by looking backward and slight restlessness. No excitation or sedation occurred.

The overall mean EP and EMG AD-related waveforms with the influence of epidurally administered methadone and the partial reversal effect of epidurally administered naloxone were determined (Figure 4). Overall statistical analysis revealed that the mean relative EP P50 RDF was affected differently by saline solution and methadone administration over time (P = 0.03). Post hoc analysis revealed that the mean relative EP P50 RDF decreased significantly following both saline solution (P = 0.008) and methadone (P < 0.001) injection. However, the decrease of the mean relative EP P50 RDF was significantly (P = 0.011) greater following methadone administration than following saline solution administration at t2.

Figure 4—
Figure 4—

Mean (thick lines in A and C) + SEM (thin lines in A and C) methadone response characteristics in 7 ponies. A—Evoked potential readings. B—Relative RDF of EP P50. C—Electromyogram readings. D—Relative RDF of N62 RDF after epidural administration of methadone (0.5 mg/kg) and naloxone (0.04 to 0.06 mg/kg) or saline solution. BL = Baseline. t1 = Time points 5 to 20 minutes after administration of methadone or saline solution. t2 = Time points 25 to 40 minutes after administration of methadone or saline solution. t3 = Time points 45 to 60 minutes after administration of methadone or saline solution. t4 = Time points 65 to 80 minutes after administration of methadone or saline solution. At t3, naloxone (methadone group) or saline solution (saline group) was administered. *Significant (P < 0.05) difference between groups. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.813

Overall statistical analysis revealed that the mean relative EMG N62 RDF was affected differently by saline solution and methadone administration over time (P = 0.035). Post hoc analysis revealed that the mean relative EMG N62 RDF significantly decreased following both saline solution (P = 0.006) and methadone (P < 0.001) injection. However, the decrease of the mean relative EMG N62 RDF was significantly greater following methadone than following saline solution at t1 (P = 0.032) and t2 (P = 0.018).

Effect of systemically administered hypnotic agents—Administration of a loading dose plus constant rate infusion of propofol led to standing sedation in all ponies. While remaining in a standing position, they were physically supported by a sling. The NWR disappeared, and statistical analysis revealed that the mean relative RDF of the EP P50 (P = 0.001) and the EMG N62 (P < 0.001) decreased significantly (Figure 5).

Figure 5—
Figure 5—

Mean (thick lines in A and C) + SEM (thin lines in A and C) propofol-response characteristics in 4 ponies. A—Evoked potential readings. Y-axis indicates stimulus intensity (mA). B—Relative RDF of EP P50. C—Electromyogram readings. Y-axis indicates stimulus intensity (mA). D—Relative RDF of EMG N62 during administration of propofol (0.5 mg/kg loading dose and 0.1 mg/kg/min constant rate infusion). See Figures 3 and 4 for remainder of key.

Citation: American Journal of Veterinary Research 70, 7; 10.2460/ajvr.70.7.813

Discussion

In the present study, EPs were characterized in ponies and subsequently evaluated for their applicability to quantifiably determine epidural antinociception in equidae. On the basis of the CV determined in this model, the EP P50 component was determined to be primarily related to nociceptive AD-fiber activity. Furthermore, the EP P50 increased with a stepwise increase in stimulus intensity and decreased following epidural methadone administration. These combined results strongly suggest the EP P50 to be quantitatively related to caudal nociception in equidae, thereby consequently proposing the EP P50 to be used as a valid quantitative method for determining the level of analgesia, compared with the more traditionally applied reflex. However, although the EP waveform characteristics (peak latency and general waveform) differed from the EMG waveform, the presence of a considerable propofol effect on the EPs prohibits the definitive exclusion of EMG or ventral horn interference in the EPs. The combined results suggest that the stimulation and recording setup described in this report allows discrimination between nociceptive and other somatosensory responses. However, it ultimately remains undetermined whether EPs are of specific neural rather than muscular origin.

To use EPs for quantifying epidural antinociception, by definition the EP must be nociception-related, that is, mediated by peripheral afferent fibers such as AD- and C-fibers.25 The fiber type mediating a specific response can be determined by calculating its CV. With a mean distance of 1 m between the stimulation and recording site in the present study and considering a peripheral CV of 15 to 35 m/s for AD-fibers, < 15 m/s for C-fibers, and 35 to 75 m/s for AA/B-fibers13,26; EP components from 28 to 67 milliseconds, such as the present EP P50, can be considered AD-fiber–related. In the present study, the results obtained with this approach indicated that the EP P50 was mediated by AD-fibers and the EP N25 was mediated by AA/B-fibers. This was further supported by the latency shifts and calculated CVs of the N25 and P50 complexes. The same is true for the early and late complex of the EMG. On the basis of the distance between the stimulation and EMG recording site, conduction velocities of AB- and AD-fibers, and a 5- to 10-millisecond delay of ventral horn efferent fibers to muscles,9 the EMG P27 and N62 was ascribed to AB- and AD-mediated stimulation, respectively. This was further supported by the latency shifts of the P27 and N62 complexes, documented by a fixed recording site after both proximal and distal stimulation. These findings are consistent with findings of Spadavecchia et al,9 who described a short-latency (0 to 80 milliseconds) AB-mediated EMG response and a longer-latency (80 to 250 milliseconds) AD-mediated EMG in Warmbloods.

Taken together, although the stimulus modality used in the present study (electrical stimulation) appeared to be non-nociceptive specific, the relatively long afferent pathways (approx 1 m) allowed for a clear distinction between the different types of afferent fibers and, consequently, the evaluation of specific nociceptive (ie, primarily AD-fiber–mediated) responses after electrical stimulation. To further validate the techniques used here, we compared the effects of a M-opioid analgesic and a nonanalgesic hypnotic between the EPs and EMGs. With respect to the effect of M-opioid analgesics, M-opioid receptors are primarily presynaptically located on the primary afferent fibers and in the neurites of dorsal root ganglion cells27 and, to a lesser degree, postsynaptically in the different layers of the dorsal horn of the spinal cord.28–30 Although M-opioids are considered to primarily affect nociceptive AD-/C- rather than tactile AB-afferent fibers,31 methadone in the present study decreased both the AD-related EP P50/EMG N62 and the AB-related EP N25/EMG P27. Similar findings were described by Carpenter et al32 and Clarke et al,33 who determined that opioids inhibit AB-, AD-, and C-fiber evoked responses. Differences in selectivity for AD-/C- and AB-afferent fibers as reported in the literature may be explained by differences in doses or route of administration as well as by species differences. In the present study, the high dose of methadone (0.5 mg/kg) may have led to high opioid concentrations in the dorsal horn and, thus, may have accounted for the effect on both nociceptive-(AD) and tactile-(AB) mediated sensory transmission. The fact that following epidural saline solution injection, both the RDF EP and RDF EMG changed can be explained by habituation to the repeated electrical stimulation.

Besides validation with the application of opioids, we also used the nonanalgesic hypnotic propofol for additional validation studies. Propofol primarily suppresses ventral horn spinal cord neurons rather than dorsal horn spinal cord neurons.12,34 Therefore, during application of relatively low doses of propofol, absence of the NWR relying on afferent dorsal horn activation and subsequent ventral horn motor activation could mistakenly be interpreted as antinociception, although nociceptive dorsal horn activity and subsequent nociceptive brain center activation can remain partially intact.11,12 The same has been reported for inhalation anesthetics, like isoflurane and halothane.11,35 In this line of reasoning, in the present study, we expected EMG to be decreased or even absent, whereas EPs were expected to be intact following propofol administration. However, both EMG and EP decreased to similar values following propofol administration. From these combined results, we concluded that the EP did not definitively reflect solely afferent dorsal horn activity. A possible explanation could be found in the location of the epidural electrode. During advancement of the epidural electrode, the measuring tip could have been translocated from a dorsal position to a more ventral position. Therefore, the disadvantage of the caudal epidural placement of the electrode is found in the uncertainty regarding its ultimate location, both in relation to the lumbosacral junction and in relation to the dorsal horn section of the spinal cord. In addition, both the active (epidural electrode) and the reference electrode were located close to the lumbosacral epaxial muscles (longissimus dorsi) of which activity might have interfered with the EP signals from the epidural space as well.

Although the stimulation setup reported here allows the successful discrimination between nociceptive and other somatosensory responses, additional research is needed for further optimization of the recording configuration to obtain clean (ie, EMG-free) dorsal horn neural activity. This would contribute to the development of a valid and quantitative measure of spinal nociceptive processing for the assessment of new analgesics or improvement of analgesia protocols in equidae.

ABBREVIATIONS

CV

Conduction velocity

EMG

Electromyogram

EP

Evoked potential

NWR

Nociceptive withdrawal reflex

RDF

Rate dispersion factor

RM

Repeated measurements

a.

Lidocaine HCl 2%, B. Braun, Melsungen, Germany.

b.

Perifix 402 filter set, B. Braun, Melsungen, Germany.

c.

Stimulator Model S88, Grass Medical Instruments, Quincy, Mass.

d.

Model SUI5, Grass Medical Instruments, Quincy, Mass.

e.

Model CCU 1A, Grass Medical Instruments, Quincy, Mass.

f.

Bio electric amplifier AB 601 G, Nihon Kohden, Tokyo, Japan.

g.

PCI 6251, Labview, National Instruments, Woerden, The Netherlands.

h.

Methadon HCl, Eurovet Animal Health, Bladel, The Netherlands.

i.

Naloxon HCl, University Pharmacy, Utrecht, The Netherlands.

j.

Propovet, Abbott Animal Health, Zwolle, The Netherlands.

k.

Benzylpenicilline Natrium, Eurovet Animal Health, Bladel, The Netherlands.

l.

Gentamycine 5%, Eurovet Animal Health, Bladel, The Netherlands.

m.

Bedozane, Eurovet Animal Health, Bladel, The Netherlands.

n.

SPSS, version 12.0, SPSS Inc, Chicago, Ill.

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Contributor Notes

The authors thank Kuei-Ming Chang, Marjon van der Kraats, and Machteld van Dierendonck for technical assistance.

Address correspondence to Dr. van Loon.