The NWR evoked by single electrical transcutaneous stimulations for the forelimb and hind limb in conscious dogs has been proposed as a new experimental method to investigate and quantify nociception.1 Studies in cats, horses,2-4 and humans5-7 have revealed that application of repeated electrical stimulations results in facilitation of the NWR,8,9 which is most likely attributable to the temporal summation of the action potentials at the level of the spinal dorsal horn neurons. Clinically, it is accompanied by an amplified sensation of pain.10 In neurophysiologic experimental settings, repetition of a fixed stimulus at low frequency results in an augmented action potential discharge of dorsal horn neurons followed by afterdischarge and increased sensitivity; this activity-dependent facilitation is called windup.11 Windup is capable of inducing the initial plastic changes at the spinal cord level leading to the development of chronic pain states.7,12 Temporal summation is involved in the early phase of windup13 in animals and has thus been widely used as an experimental model of central excitability. In animal research, the use of experimentally induced windup and chronic pain by injection of chemical agents14 or repeated electrical stimuli15,16 has been limited to rats and cats that have been anesthetized or undergone spinal cord transection, with the drawback of the invasiveness of those techniques. Dogs affected with orthopedic diseases such as hip or elbow joint dysplasia17 or spondylarthrosis, cancer, or oral cavity conditions18 can develop signs of chronic pain, and both veterinarians and owners are convinced that those dogs should receive adequate pain-relieving treatment. However, accurate detection of signs of pain is difficult and relies on subjective description of pathologic locomotion or behavior and demeanor patterns or use of composite pain scales. In dogs, only a few scales are reported to be valid for evaluating chronic osteoarthritis-associated pain,17,19 and other types of chronic pain are actually not addressed. Therefore, it would be of major interest to have an objective, noninvasive technique with which the degree of sensory dysfunction and the effectiveness of analgesic treatments in these canine patients could be assessed quantitatively, thereby improving the quality of available treatments. In horses, use of NWR assessment as an experimental method to assess temporal summation has been validated20 and it is sufficiently sensitive to be used in the investigation of analgesic drug efficacies.3 By use of a previously developed neurophysiologic technique for quantification of nociception in dogs,1 the purpose of the study reported here was to investigate the facilitation of the NWR by repeated electrical stimuli and the associated behavioral response scores in conscious, nonmedicated dogs as a measure of temporal summation and analyze the influence of stimulus intensity and frequency on temporal summation responses. Our hypotheses were that in conscious, nonmedicated dogs, repetitive electrical stimulations of the forelimb and hind limb would facilitate the NWR, that this facilitation could be quantified as a measure of temporal summation, and that frequency and intensity of the stimulation would influence temporal summation threshold and its characteristics.
Materials and Methods
Animals—Eight adult male purpose-bred Beagles (mean body weight, 9.1 ± 1.7 kg; age, 1.5 to 5 years) were included in the study. They were judged to be healthy on the basis of findings of physical examination and clinicopathologic analyses. Dogs were housed together in runs (10 dogs/run), and food was withheld in the morning prior to the experimental session. The experiments were approved by the Committee for Animal Experimentation of the Canton Basel City, Switzerland (approval No. 2090).
Experimental procedure—Measurements were begun in the morning and took place in a constant-temperature (22°C) room. Prior to the experiment, each dog underwent physical examination. The stimulation and recording sites were clipped, shaved, and degreased. The dog was placed in right lateral recumbency in a comfortable, commercial dog bed (filled with corncob balls) that took the shape of the body. The limbs were extended laterally in a natural position, but not supported, and without weight bearing or movement restriction of the nondependent limb. Typically, the dog accepted to lie still. The surface electrodes were then positioned, the nerves were transcutaneously stimulated by electrical stimuli, and the response was recorded by surface EMG. On completion of the trials, the electrodes were removed; to avoid local reactions, the skin was washed and a dermatologic creama was applied.
Recording and stimulation equipment—Self-adhesive stimulation electrodesb were applied over the dorsal branch of the ulnar nerve at the level of the left fifth metacarpal bone of the forelimb and over the lateral plantar digital nerve at the level of the fourth metatarsal bone of the hind limb. In both limbs, the 2 electrodes were positioned just distal to the base and proximal to the head of each bone. The electrodes were placed parallel to the nerve with the anode in distal position, and interelectrode distance was 0.8 cm. The distal portion of each limb was bandaged to prevent dislocation of the electrodes.
By means of pairs of self-adhesive electrodes,b surface EMGs were recorded from the deltoideus and cleidobrachialis muscles of the forelimb and from the caput pelvis of the biceps femoris muscle and the cranial tibial muscle of the hind limb. Special care was taken to place the electrodes over the muscle bellies at a distance of 1 cm to avoid multichannel cross-talk contamination from adjacent muscles, thereby minimizing commonmode noise and stimulus artefacts. The ground electrodec was placed over the plantar side of the right hind foot and taped in place. Flexible leads were connected to the electrodes. The resistance of each electrode pair was checked and confirmed to be < 5 kΩ before the start and at the end of each experimental session.21
Stimulation and recordings were performed by use of a specially designed computerized system. The final stage of the stimulator that received input from the computer was a battery-powered optoisolated constant-current device with a maximum voltage of 100 V and a maximal current of 40 mA. Electromyographic signals were amplified with an overall gain of 5,000 and bandpass of 7 to 200 Hz (first-order active filters with 6 dB/octave slope). They were passed through a digital converter to a computer for further processing and storage.
Electrical stimulations—The stimulus consisted of standard,5,22,23 constant-current, square-wave–pulse, train-of-five, 1-millisecond pulses delivered at 200 Hz (equivalent to an ISI of 5 milliseconds). The experimental session was started with the determination of It value for each dog. The initial intensity was 1 mA1; if no reflex response could be elicited, the current was gradually increased in steps of 0.2 mA until It was determined. The It was defined as the minimum stimulus intensity that evoked a reflex response (that was repeatable 3 times) from the deltoideus muscle (forelimb) and the biceps femoris muscle (hind limb) in the 20- to 100-millisecond poststimulation epoch that had an amplitude > 10 times the EMG background activity and duration > 10 milliseconds and that elicited a behavioral reaction scored as 1 or 2.1 The 100-millisecond prestimulation interval and an interval of 400 milliseconds after stimulation were analyzed (512 sampling points; sampling frequency, 1,280 Hz).
Immediately after the It was defined, the temporal summation threshold was determined. The study design combined different frequencies and number of stimuli4,5 so that the total duration of the train was constant because time is essential when integration over time is to be studied. The total stimulation time was 2 seconds.4,5 Three stimulation frequencies were used in random order: 2 Hz (ISI, 500 milliseconds) with 4 pulse trains delivered over the 2-second period, 5 Hz (ISI, 200 milliseconds) with 10 pulse trains delivered over the 2-second period, or 20 Hz (ISI, 50 milliseconds) with 40 pulse trains delivered over the 2-second period. The stimulation intensities were fractions of the individual It (0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 1.1 × It) and were applied in a stepped manner to avoid possible startle reactions. Sufficient time was allowed between stimuli to avoid habituation,24,25 and care was taken that the limbs of each dog were extended without spontaneous movements or tremors, factors which could affect the EMG recordings.26,27 All the combinations of stimulus intensity and frequency were applied only once to limit the duration of the experiment and any discomfort for the dogs. The 0- to 500-millisecond epoch prestimulation interval, the 500- to 2,500-millisecond epoch stimulation interval, and finally, the 2,500- to 4,000-millsecond epoch poststimulation interval were analyzed (sampling frequency, 1 kHz).
Each experimental session was started with the hind limb (session I), and afterwards, the whole procedure was repeated for the forelimb (session II). The interval between sessions was 1 hour, during which dogs were allowed to stand and walk.
Behavioral response to repeated stimulations— The same investigator (AB) observed and scored the behavioral reactions of the dogs to each stimulation. The investigator was unaware of the timing, intensity, and frequency of the stimulus applied. The behavioral score was assigned as follows: 0 = no movement; 1 = muscle fasciculation; 2 = flexion of elbow or stifle joint followed by relaxation; 3 = flexion of entire fore- or hind limb followed by relaxation; 4 = flexion of fore- or hind limb, and flexion maintained; 5= sustained flexion of fore- or hind limb and general awareness (ie, extension or shaking of the limb or attempts to stand from the lying position); and 6 = fore- or hind limb flexion, general awareness, and vocalization.
Reflex analysis after repeated stimulations—Not including the stimuli used to determine It, 96 EMG responses (8 intensities and 3 frequencies for 4 muscles) were analyzed off-line for each dog. The temporal summation threshold was defined as the intensity at which the root-mean-square amplitude of the EMG signal in the 20- to 100-millisecond interval increased and exceeded 10 times the background activity in the third or fourth stimulus of the pulse train and was associated with a clear behavioral reaction scored as ≥ 2. The latency to onset of the NWR evoked by the first pulse train in the series was measured.
To quantify the magnitude of the reflex response elicited by the repeated stimulations and minimize interindividual variability, relative amplitude was calculated as the ratio between the mean root-mean-square reflex activity of each 20- to 100-millisecond (50 milliseconds for 20 Hz) poststimulation interval in the 500- to 2,500-millisecond epoch and the root-mean-square background activity in the 0- to 500-millisecond epoch.
Data analysis—Nonparametric tests were used because of the small sample size. Results are expressed as median and 25% to 75% interquartile range. The intensity required to elicit temporal summation, latency, relative amplitude of the reflex, and behavioral response score at temporal summation intensity at each frequency were compared by use of the Friedman test, followed by a Tukey test if needed; the same test was used to analyze the latency, the relative amplitude of the reflex for each of the 4 muscles, and the behavioral scores elicited by graded stimulations at each frequency.
For each frequency, the Wilcoxon signed rank test was used to compare temporal summation thresholds and behavioral scores between forelimb and hind limb. Correlations between stimulation intensities, behavioral response scores, and the relative amplitude of the reflex response were calculated for each stimulus frequency by use of the Spearman rank test. Significance was set at a value of P < 0.05.d,e
Results
No abnormalities were detected on physical examination of the dogs. All dogs tolerated the experiment well; throughout the study period, they were relaxed and remained in lateral recumbency. None of the dogs vocalized at any of the stimulus intensities used, and the behavioral reactions ceased on stimulus cessation. The day after the experiments, some erythema of the skin on the lateral metacarpal and tarsal region was present but healed fully. No short- or long-term (6-month follow-up) skin damage was noticeable.
Individual NWR threshold—Median It was 2.1 mA (range; 25% to 75% interquartile interval=, 1.7 to 2.9 mA) for the hind limb and 2.5 mA (range, 2.0 to 3.6 mA) for the forelimb. There was no significant (P = 0.563) difference in median It between limbs.
Temporal summation threshold—For the hind limb, temporal summation occurred at 0.65 × It (range, 0.45 to 0.7 × It), 0.6 × It (range, 0.45 to 0.75 × It), and 0.5 × It (range, 0.5 to 0.6 × It) at 2, 5, and 20 Hz, respectively; these It fractions were significantly (P = 0.001) lower than It, indicating that the NWR in dogs is facilitated via application of repeated stimulations (Figure 1). Frequency had no significant (P = 0.794) impact on the temporal summation thresholds.
Representative rectified EMGs obtained from the biceps femoris (BF) and cranial tibial (CT) muscles with a repeated stimulus at 0.7 × It intensity at 2-, 5-, and 20-Hz stimulus frequencies in a conscious, nonmedicated dog. The x-axis represents time in milliseconds, and the y-axis represents amplitude of the reflex in microvolts. The 500- to 2,500-millisecond stimulation epoch is delineated by the vertical lines. Notice that the magnitude of summation differs among frequencies and between muscles.
Citation: American Journal of Veterinary Research 68, 8; 10.2460/ajvr.68.8.899
For the forelimb, temporal summation occurred at 0.85 × It (range, 0.7 to 0.9 × It), 0.9 × It (range, 0.75 to 1 × It), and 0.75 × It (range, 0.65 to 0.85 × It) for 2, 5, and 20 Hz, respectively; these It fractions were significantly (P = 0.008) lower than It, except for the 5 Hz when corrected for multiple testing. The intensities needed to elicit temporal summation were significantly lower for the hind limb, compared with the forelimb, for all frequencies (2 Hz, P = 0.008; 5 Hz, P = 0.039; and 20 Hz, P = 0.016).
The latencies of the reflex at temporal summation intensity for the biceps femoris muscle were 2,036, 1,332, and 700 milliseconds and for the deltoid muscle were 2,036, 1,332, and 753 milliseconds at 2, 5, and 20 Hz, respectively. Reflexes appeared significantly (P < 0.001) earlier in the stimulation epoch with higher frequencies. There was a significant negative correlation between latency and stimulus intensity for the biceps femoris muscle at 5 (r = −0.62; P < 0.001) and 20 Hz (r = −0.51; P < 0.001) and for the deltoid muscle at 2 (r = −0.38; P = 0.02) and 5 Hz (r = −0.4; P = 0.02).
At 20 Hz, the relative amplitude of the reflex was significantly lower for the biceps femoris (P = 0.002) and deltoideus (P = 0.03) muscles at temporal summation threshold intensity, compared with values at 2 and 5 Hz. By increasing stimulation intensity, the relative amplitude of the reflexes for the 4 muscles increased significantly for all frequencies (Table 1). Overall, the relative amplitude of the reflex recorded for the biceps femoris muscle was significantly (P < 0.001) higher than that of the deltoideus muscle, independent of the frequency. It was a general characteristic among all dogs and for all frequencies that the median root-mean-square values in the 20- to 100-millisecond (20- to 50-millisecond for the 20-Hz frequency) poststimulation interval of each NWR in a stimulus series progressively increased after the third or fourth stimulus (Figure 2). Thereafter, when the 20-Hz frequency was used, the root-mean-square of the single NWRs significantly (P < 0.001) decreased, compared with the previous amplified NWRs, indicative of habituation or inhibition. This phenomenon occurred for the 2 and 5 Hz only at suprathreshold intensities.
Median values (25% to 75% interquartile range) of the relative amplitude of the reflex evoked by increasing stimulation intensities (fractions of the individual It) and the behavioral response scores at frequencies of 2, 5, and 20 Hz applied to the biceps femoris (BF), cranial tibial (CT), deltoideus (D), and cleidobrachialis (CB) muscles in 8 conscious, nonmedicated dogs. Values of P were derived via a Friedman repeated-measures ANOVA (significance set at P < 0.05) for comparisons of stimulus intensities within a row.
| Frequency (Hz) | Variable | Stimulus intensity (X It) | P value | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0.4 | 0.5 | 0.6 | 0.7 | 0.8 | 0.9 | 1.0 | 1.1 | |||
| 2 | Behavioral scores for hind limb | 0.5 (0–2.0) | 1.0 (0.5–2.5) | 2.0 (0.25–2.0) | 2.0 (2.0–3.75) | 2.0 (2.0–2.75) | 3.0 (2.25–3.0) | 3.0 (3.0–3.5) | 3.0 (3.0–4.0) | < 0.001 |
| Relative amplitude | ||||||||||
| BF | 5.8 (1.2–32.1) | 6.7 (2.6–61.7) | 8.2 (3.2–22) | 23.3 (16.9–38.1) | 43.9 (19.1–50.0) | 26.7 (15.8–54.5) | 41.2 (20.8–50.4) | 51.1 (33.6–76.3) | 0.009 | |
| CT | 2.1 (1.0–4.6) | 2.5 (1.1–9.9) | 2.3 (1.4–3.0) | 5.6 (4.6–14.0) | 10.0 (4.3–16.9) | 9.7 (5.8–13.6) | 12.2 (10.6–21.0) | 13.5 (10.8–25.7) | < 0.001 | |
| Behavioral scores for forelimb | 0.0 (0.0–0.0) | 0.0 (0.0–1.0) | 1.0 (0.5–1.5) | 2.0 (1.0–2.0) | 2.0 (1.5–2.5) | 2.0 (1.5–3.0) | 3.0 (2.0–3.0) | 3.0 (3.0–3.0) | < 0.001 | |
| Relative amplitude | ||||||||||
| D | 1.0 (1.1–2.4) | 3.0 (1.4–4.9) | 5.0 (1.5–6) | 6.2 (4.9–7.8) | 9.5 (6.4–16.2) | 12.4 (8.6–18.9) | 9.6 (6.9–18.0) | 11.3 (9.4–19.6) | 0.007 | |
| CB | 2.5 (1.8–3.6) | 2.8 (1.9–5.9) | 4.8 (1.9–7.3) | 9.1 (3.6–16.0) | 9.3 (6.6–16.2) | 10.3 (8.1–20.7) | 16.6 (12.1–23.7) | 13.7 (9.1–27.7) | < 0.001 | |
| 5 | Behavioral scores for hind limb | 0.0 (0.0–1.5) | 1.0 (0.5–1.5) | 1.0 (0.25–2.0) | 2.0 (1.0–3.0) | 2.5 (2.0–3.0) | 3.0 (3.0–3.5) | 3.0 (3.0–4.0) | 4.0 (3.0–4.0) | < 0.001 |
| Relative amplitude | ||||||||||
| BF | 2.3 (1.2–8.7) | 8.9 (4.7–15.6) | 6.1 (4.2–18.5) | 17.2 (6.2–36.8) | 13.5 (10.3–28.8) | 34.1 (29–58.6) | 52.3 (35.8–60.2) | 50.1 (42.4–80.1) | < 0.001 | |
| CT | 2.3 (1.3–6.3) | 2.6 (1.1–4.7) | 1.7 (1–2.3) | 6.0 (3.3–10) | 6.1 (3.2–12.6) | 11.7 (6.1–14.1) | 14.5 (8.7–18.1) | 18.2 (13.6–20.6) | < 0.001 | |
| Behavioral scores for forlimb | 0.0 (0.0–0.0) | 0.0 (0.0–0.5) | 1.0 (0.0–1.0) | 1.0 (0.0–2.0) | 1.5 (1.0–2.5) | 2.0 (2.0–2.5) | 2.5 (2.0–3.0) | 3.0 (3.0–3.5) | < 0.001 | |
| Relative amplitude | ||||||||||
| D | 1.2 (0.9–1.5) | 1.2 (1.0–1.3) | 1.9 (1.5–3.2) | 4.5 (1.9–5.8) | 5.6 (2.2–7.9) | 10.1 (6.5–21.3) | 15.1 (7.8–15.5) | 17.9 (11.1–25.0) | < 0.001 | |
| CB | 1.8 (1.7–2.8) | 2.1 (1.2–4.5) | 4.2 (2.5–12.2) | 7.2 (2.7–20.9) | 10 (8.2–15.2) | 14.2 (11.0–17.0) | 20 (12.4–42.9) | 25 (21.4–30.7) | < 0.001 | |
| 20 | Behavioral scores for hind limb | 0.5 (0.0–1.5) | 2.0 (0.5–2.5) | 2.0 (2.0–3.0) | 3.0 (2.0–3.0) | 3.0 (3.0–3.5) | 3.0 (3.0–3.75) | 3.0 (3.0–3.0) | 4.0 (3.0–4.0) | < 0.001 |
| Relative amplitude | ||||||||||
| BF | 3.8 (1.4–6.6) | 6.6 (4.1–10.8) | 15.0 (7.9–23.1) | 23.0 (18.6–31.0) | 19.7 (15.4–28.8) | 22.1 (14.3–37.1) | 18.5 (15.4–35.9) | 36.3 (20.4–49.3) | < 0.001 | |
| CT | 1.2 (1.0–2.8) | 6.0 (3.4–7.2) | 5.9 (0.6–11.5) | 6.4 (4.1–10.6) | 7.3 (6.5–14.7) | 12.7 (3.7–15.1) | 9.9 (5.3–19.1) | 12.3 (7.3–16.2) 0.189 | (7.3–16.2) | |
| Behavioral scores for forlimb | 0.0 (0.0–0.0) | 0.0 (0.0–1.0) | 1.0 (0.0–1.0) | 1.0 (0.0–1.0) | 2.0 (1.0–2.0) | 2.0 (1.5–3.0) | 3.0 (2.5–3.5) | 3.0 (3.0–4.0) | < 0.001 | |
| Relative amplitude | ||||||||||
| D | 1.3 (1.1–1.9) | 1.7 (0.8–4.0) | 1.9 (1.5–4.2) | 2.8 (1.4–4.3) | 4.7 (3.1–7.5) | 6.5 (2.8–22.7) | 8.6 (5.3–19.7) | 7.3 (4.4–19.3) | < 0.001 | |
| CB | 2.2 (1.1–3.8) | 3.3 (1.9–5.8) | 2.0 (1.7–3.0) | 4.2 (2.6–9.3) | 6.8 (3.5–10.0) | 18.1 (5.5–30.3) | 12.7 (7.9–25.0) | 16.5 (8.7–32.7) | < 0.001 | |
Median values of the root-mean-square amplitude calculated in the 20- to 100-millisecond poststimulation intervals at 2- (A and D) and 5-Hz (B and E) frequencies and in the 20- to 50-millisecond poststimulation intervals at the 20-Hz (C and F) frequency of each NWR in a stimulus series performed in the biceps femoris (A through C) and deltoideus (D through F) muscles of 8 dogs. In the 2-second stimulation interval, 4, 10, and 40 reflexes were evoked for the 2-, 5-, and 20-Hz frequencies, respectively. At each stimulus frequency, 8 intensities were used (0.4 to 1.1 × the individual It).
Citation: American Journal of Veterinary Research 68, 8; 10.2460/ajvr.68.8.899
During the 2,500- to 4,000-millisecond poststimulation epoch, distinct muscular activity of the biceps femoris and the cranial tibial muscles of the hind limb was recorded only at the higher intensities of 1.0 and 1.1 × It at 2 and 20 Hz (P = 0.002 and P < 0.001, respectively) and at 0.9 × It when 5 Hz was used (P < 0.001). In the same epoch, the muscular activity was negligible for both the deltoideus and cleidobrachialis muscles of the forelimb, except at intensities of 1.0 and 1.1 × It at 5 Hz (P < 0.001 and P = 0.003, respectively).
At subthreshold intensities, the initial behavioral response of dogs was a localized fasciculation of the flexor muscles. At temporal summation threshold intensity, there was a slow flexion of the elbow or stifle joint, without complete closure of the joint angle, immediately followed by relaxation at cession of the repeated stimulus. With increasing stimulus intensity, the behavioral response was complete flexion of the entire limb and the muscular contraction was maintained. With the highest intensities (0.9 to 1.1 × It), the limb was firmly flexed close to the body and then extended cranially for the forelimb or caudally for the hind limb (mimicking an attempt to eliminate an unpleasant stimulus to the foot). Behavioral response scores at temporal summation intensity for the hind limb were 2 (range, 2 to 2.5), and there was no significant difference in frequencies for hind (P = 0.654) and forelimbs (P = 0.967). With increasing stimulation intensities, behavioral scores increased significantly (P < 0.001) for all frequencies (Table 1). For the hind limb, the 20-Hz frequency elicited the highest behavioral scores, compared with the 2- and 5-Hz frequencies (P = 0.003). For the forelimb, there were no differences in behavioral scores among frequencies. Overall, the behavioral scores were significantly (P < 0.001) higher for the hind limb than the forelimb, independent of the frequency.
Analyses revealed a significant positive correlation between the stimulation intensity (P < 0.001) and the relative amplitude of the reflex (P < 0.001) and also between the stimulation intensity and the behavioral reaction scores for the hind limb at each frequency (P < 0.001). The relative amplitude also correlated positively with the behavioral reaction scores (P < 0.001). The highest correlation coefficients were obtained at 5 Hz in both the fore- and hind limb (Table 2).
Correlations between the stimulus intensity and both the relative amplitude of the reflex and the behavioral scores and between behavioral scores and relative amplitude of the reflex for the biceps femoris and cranial tibial muscles of the hind limb and the deltoideus and cleidobrachialis muscles of the forelimb of 8 conscious, nonmedicated dogs. Correlations were calculated by use of a Spearman rank test (significance set at a value of P < 0.05). Values of P were < 0.001 unless stated otherwise.
| Variable | Frequency (Hz) | Behavioral score | Relative amplitude | ||||||
|---|---|---|---|---|---|---|---|---|---|
| BF | CT | D | CB | BF | CT | D | CB | ||
| Intensity (X It) | 2 | 0.63 | 0.64 | 0.82 | 0.83 | 0.51 | 0.63 | 0.65 | 0.60 |
| 5 | 0.8 | 0.27 (P = 0.03) | 0.8 | 0.80 | 0.78 | 0.7 | 0.76 | 0.73 | |
| 20 | 0.53 | 0.74 | 0.82 | 0.81 | 0.63 | 0.5 | 0.62 | 0.60 | |
| Behavioral score | 2 | NA | NA | NA | NA | 0.63 | 0.67 | 0.83 | 0.80 |
| 5 | NA | NA | NA | NA | 0.77 | 0.15 (P = 0.22) | 0.79 | 0.88 | |
| 20 | NA | NA | NA | NA | 0.53 | 0.63 | 0.75 | 0.75 | |
NA = Not applicable.
See Table 1 for key.
Discussion
Results of the present study have indicated that temporal summation could be elicited and assessed in conscious, nonmedicated dogs. Repetition of electrical stimuli of subthreshold intensity, which alone did not evoke a withdrawal reflex, summated and facilitated the NWR in both fore- and hind limbs, and lower intensities were effective for the hind limb, compared with the forelimb. In dogs, the facilitation of the reflex does not seem to depend on frequency of stimulation, at least at the frequencies used in the study reported here. To our knowledge, the effect of repeating low-intensity stimuli at different frequencies in dogs has not been previously investigated. In the present study, a previously developed method of assessing the NWR elicited by single electrical stimulations to quantify nociceptive processes in dogs1 was implemented and revealed that the intensity of stimulation had a significant effect on facilitation. We believe that the data obtained in the study reported here deepen the understanding of central integration of somatosensory processing in canids.
In humans, application of repeated electrical stimulations enables facilitation of the NWR2-9,11,28,29 as a result of the temporal summation of action potentials at the level of the spinal dorsal horn neurons. Temporal summation is believed to reflect the early phase of windup, which is part of the pathophysiologic basis of clinical persistent pain syndromes.7,12 Therefore, in humans, the psychophysical and electrophysiologic responses to repetitive nociceptive stimulations have been assessed as a noninvasive experimental surrogate of windup.30 Recent research15 has revealed that windup is only the initial step of the long-lasting state of neuronal hyperexcitability and plastic changes that develop during central sensitization. Nevertheless, temporal summation remains a most interesting experimental tool with which to investigate the degree of sensorial dysfunction and evaluate the analgesic efficacy of drugs in experimental and clinical settings in humans31 and other animals.2-4,6,12,29,32,33
The temporal summation threshold definition used in the present study in dogs was based on review of the human medical literature, in which various definitions have been proposed.5,10,32,34-36,f Among those reports, the increase in amplitude of the last 1 or 2 reflexes above a certain limit is considered indicative of facilitation. To assess the temporal summation by the size of 1 reflex response only would be too sensitive to the natural variation in reflex amplitude and possible technical artifacts.
The mechanisms of temporal and spatial summation involve wide dynamic range neurons37 located in the dorsal horn, which receive afferent information from AΔ and C fibers. Summation of afferent activity seems to be more pronounced for C fibers that mediate second pain sensations, compared with AΔ fibers that mediate first pain sensations.6,29,38 Many studies5,7,34,36 of temporal summation in humans have concentrated on the facilitation of the NWR reflex that is mediated by AΔ fibers. Activation of AΔ fibers causes a central discharge that has a duration of several hundred milliseconds,39 which can explain why repeated nociceptive electrical stimuli result in facilitated polysynaptic reflexes. In the present study, the analysis of the reflex activity focused on the AΔ fiber–evoked activity expressed in the 20- to 100-millisecond poststimulation intervals.1 The reflex activity attributable to C fibers would appear later, from 250 to 830 milliseconds after each stimulus.40-42 In further studies in dogs, it would be interesting to differentiate the contribution of AD and C fibers in the evoked responses by use of selective fiber blockade.
Behavioral responses to repeated stimulations were used as a psychophysical correlate of the dogs' perception of the electrical nociceptive stimuli. The behavioral pattern was quite stereotyped and differed from the behavioral reactions observed when a single stimulus was applied1; for example, localized muscle fasciculations with a repeated stimulus at subthreshold intensity were never detected after application of a single stimulus. At temporal summation intensity, the entire limb was flexed, whereas only localized joint flexion was induced with a single stimulus; an interpretation for this is that the nociceptive impulse is perceived more intensely despite the lower intensity, in accordance with findings in humans.5,7,10,29
Facilitation of the NWR for the hind limb occurred at intensities that were significantly lower than It, whereas for NWR facilitation in the forelimb, intensities close to It were needed; at the same current intensity, the behavioral scores were lower for the forelimb than the hind limb. The reason for this remains unclear. In humans, few investigations26,43 have analyzed the forearm NWR. In horses, the NWR and its facilitation have been studied for both fore- and hind limbs4,20 and only minor differences in the characteristics of temporal summation between the limbs were noticeable. In the present study, an interval (1 hour) was allowed between hind limb and forelimb measurements; it is unlikely that activation of diffuse noxious inhibitory control could have modulated the temporal summation44 or that dogs could have adapted or modified their level of reaction.45 Whether the spinal neuronal organization of the fore- and hind limb differs or the supraspinal modulation for the forelimb is more pronounced than that for the hind limb in dogs will require verification in future studies.
In humans, the characteristics of the reflex elicited by repeated stimuli are affected both by the frequency (frequencies ranging from 0.5 to 20 Hz) and the intensity of the stimulus used.5,7,46 In the present study, 3 frequencies were evaluated (2, 5, and 20 Hz) to ascertain which would better elicit summation in dogs. The frequencies were applied in random order, and during behavioral assessments, the investigator (AB) was unaware of which frequency was used. With increasing stimulation frequency, the number of stimuli increased but the total duration of the stimulus was kept constant in accordance with previous studies.4,5 As our study focused on the buildup in reflex response during application of the pulse train, the total number of stimuli was of little importance, whereas a fixed duration of the stimulation time was essential. Other study designs could have been used, such as combining different numbers of stimuli with a fixed frequency or different frequencies with a fixed number of pulses, all of which would have had a potential influence on the experimental outcome. As determined in horses,4 the frequencies used did not influence temporal summation thresholds in dogs. Nevertheless, there was a significant reduction in the latency of the reflex summation as the frequency of stimulation was increased, which fits with the concept of temporal summation: the greater the frequency of the stimuli, the earlier the appearance of the facilitated reflex. For both the forelimb and the hind limb, the 5-Hz frequency offered the best correlation between the stimulus intensity and the relative amplitude or the behavioral scores. In agreement with findings of studies5,7 in humans, the behavioral scores for the hind limb at the 20-Hz frequency were higher than the scores elicited with the 2-and 5-Hz frequencies, which indicates that the nociceptive stimulus was perceived as more intense; furthermore, at 20 Hz, reflex facilitation effectively dissipated5 with a significant reduction in the root-mean-square amplitude of the reflex activity during the final part of the stimulus series, compared with the other frequencies. This can be explained by habituation or activation of descending inhibitory systems.46 The intensity of stimulation affected the magnitude of the reflex response with a significant positive correlation between the stimulus intensity-response curve and the reflex amplitude-response curve; on the basis of neuronal recordings in other species, this is probably attributable to spatial summation of the afferent information at spinal level.5,16 As for the single stimulation,1 the stimulus intensity was inversely correlated to the latency of the reflex. The behavioral response scores increased with increasing stimulation intensities as an indication of increased nociception. This positive correlation between the intensity of the stimulus and the magnitude of temporal summation (as measured by the relative amplitude) and its perception (as measured by the behavioral scores) as between the relative amplitude and the behavioral response scores confirm the consistency of experimentally induced temporal summation in dogs.
As the data obtained in the present study have indicated, it is possible to elicit temporal summation in conscious, nonmedicated dogs. Temporal summation appeared to be more easily elicited from the hind limb, compared with the forelimb, but the reason for this difference remains to be elucidated. Despite that lack of influence of the stimulus frequency on temporal summation thresholds, the highest correlations between the stimulus intensity and both the relative amplitude and the behavioral reaction scores were obtained at the 5-Hz frequency, which is therefore recommended as the standard for future studies in dogs. Temporal summation can be used as a model of windup in dogs both for better understanding of the pathophysiologic processes involved in chronic pain states and to establish the efficacy of analgesic drugs specifically and objectively in pharmacodynamic studies in this species.
ABBREVIATIONS
| NWR | Nociceptive withdrawal reflex |
| EMG | Electromyography |
| ISI | Interstimulus interval |
| It | NWR threshold intensity |
Weleda Heilsalbe, Weleda AG, Arlesheim, Switzerland.
Neuroline 700 05-j, Medikotest A/S, Olstykke, Denmark.
Synapse 32 mm, Ambu A/S, Ballerup, Denmark.
SigmaStat, version 3.1, Systat Software Inc, Point Richmond, Calif.
SygmaPlot, version 9.0, Systat Software Inc, Point Richmond, Calif.
Andersen OK. Physiological and pharmacological modulation of the human nociceptive withdrawal reflex. PhD thesis, Center for Sensory-Motor Interaction, Faculty of Engineering, Science and Medicine, Aalborg University, Aalborg, Denmark, 1996;48.
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