Laminitis is an extremely painful condition of the feet in horses. The pathophysiologic mechanisms that result in laminitis remain poorly understood but appear to involve both vascular and inflammatory responses within the hoof that lead to disruption of the lamellar dermoepidermal junction, impaired biomechanical function, and substantial signs of pain.1–3 Although many signs of pain in chronically laminitic horses originate from local and systemic pathological inflammatory responses, morphological changes and upregulation of injury markers in both peripheral and central neurons indicate that there is also an important neuropathic component. Along with these neuropathic changes, an increase in offloading frequency of affected limbs and other behavioral modifications suggestive of maladaptive pain characterized by hyperalgesia and allodynia have also been identified.4 In addition, high plasma concentrations of the proinflammatory cytokine TNF-α, an important sensitizer of peripheral and central nociceptive pathways and critical mediator of maladaptive pain,5–9 have been detected in horses with chronic laminitis.10 These findings can be exploited to develop more effective analgesic treatments, thereby improving the well-being and productivity of affected horses and providing further insights into the mechanisms of this painful condition.
Mechanistically, drugs such as tramadol hydrochloride and ketamine hydrochloride are suitable candidates for multimodal analgesia in neuropathic and inflammatory pain states.11–13 Tramadol is a synthetic, centrally acting analgesic with a multimodal mechanism of action. It is both a weak opioid receptor agonist with selectivity for the μ-opioid receptor and a weak inhibitor of synaptic reuptake of norepinephrine and serotonin.14–16 Tramadol-induced analgesia may also result from modulation of inflammatory mediators such as proinflammatory cytokines and prostanoids.17,18 Tramadol (5 mg/kg) administered orally to healthy horses that have no painful conditions19 has high bioavailability and maximum concentrations much greater than the suggested therapeutic threshold in humans.20,21 When administered to horses orally at a dose of 2 mg/kg, tramadol was readily detectable in plasma in one study22 but not in another.23 The reason for this discrepancy may be due to methodological differences in measuring tramadol concentration in equine plasma or to variations in oral bioavailability at lower doses.
Ketamine is a phencyclidine derivative and is most commonly used as a dissociative anesthetic agent. At both anesthetic and subanesthetic doses, ketamine induces analgesia through numerous signaling pathways, although NMDA receptor–dependent mechanisms appear to be the most important.24 Ketamine has been shown to regulate inflammatory responses and to reduce hyperalgesia, and its administration results in almost complete remission of symptoms of refractory neuropathic pain in humans.24–32 To our knowledge, there are no reports of the effects of systemically administered ketamine on hyperalgesia in horses, although a reduction in incisional hyperalgesia associated with epidural administration of ketamine was detected in 1 study.33 Evidence from human patients and other animals with experimentally induced pain suggests that simultaneous pharmacological modulation of opioid and NMDA receptor signaling systems results in superior analgesia, compared with that achieved via modulation of either signaling system alone.34 The purpose of the study reported here was to characterize the analgesic effects of tramadol administered orally and to explore whether short-term IV administration of subanesthetic doses of ketamine could modulate tramadol-induced analgesia in horses with signs of pain associated with naturally occurring chronic laminitis.
Materials and Methods
Animals—Fifteen client-owned adult (4 to 20 years old) horses of either sex and representing several breeds with naturally occurring chronic laminitis were used in the study, which had a randomized, crossover design. Inclusion criteria included a clinical diagnosis of bilateral forelimb laminitis of at least 3 months' duration, with radiographic confirmation of displacement of the distal phalanx relative to the hoof wall in 1 or both forefeet, and evidence of a decrease in total forelimb load as previously described.35 All horses were medically evaluated for overall health through physical examination and laboratory analyses (CBC and serum biochemical analysis) prior to enrollment in the study. Horses were unshod for the duration of the study and were maintained on deeply bedded stalls (shavings) with free access to water. Horses were fed coastal hay twice daily and fed concentrate each morning. Owner consent was obtained prior to enrollment of any horse in the study. The experimental protocols were reviewed and approved by the Clinical Research Review Committee at Texas A&M University.
Drug treatments—At the time of enrollment, each horse was randomly assigned to receive 1 of 2 treatments consisting of tramadol hydrochloridea alone or tramadol hydrochloride and ketamine hydrochloride.b After an interval of at least 60 days, each horse received the other treatment.
For either treatment, tramadol tablets were crushed into powder, mixed with corn syrup,c and administered orally with a syringe. Each horse received 5 mg of tramadol/kg every 12 hours (at 8:00 am and 8:00 pm) for a period of 7 days. The dose of tramadol was selected on the basis of published information at the time the study was being designed, which indicated that bioavailability was good when tramadol was administered at a dose of 5 mg/kg19 but not good when tramadol was administered at a dose of 2 mg/kg23, and on the basis of results obtained in a pilot study involving 1 horse in which doses of 2.5, 5, and 10 mg of tramadol/kg were tested. In that horse improvement in off-loading frequency was not detected with the lowest dose, was detectable with the midrange dose, and was not enhanced further with the highest dose. Therefore, a dose of 5 mg of tramadol/kg was selected for the experiments. The corn syrupc used was selected because it was frequently used to dilute other medications for oral administration to horses in our facility. The oral route was selected, instead of administration directly into the stomach via nasogastric tube, because of its clinical relevance and to avoid possible complications associated with repeated nasogastric tube placements in these client-owned horses.
Before the start of each tramadol-ketamine treatment experiment, a 14-gauge, 5.25-inch-long IV catheterd was placed in 1 jugular vein for ketamine administration. The catheter remained in place for the duration of ketamine administration (3 days) and was inspected at least 3 times/d for signs of adverse reactions (ie, infection, inflammation, or thrombophlebitis). During the initial 3 days of tramadol administration, ketamine was administered at 0.6 mg/kg/h for 6 h/d (from 8:00 am to 2:00 pm) with an automated syringe pumpe that was attached to the horse's neck with a loose bandage. The dose and duration of ketamine were selected so that the treatment protocol could be safely applied for these client-owned animals36,37 and would be sufficient to prevent and possibly revoke NMDA-dependent changes in gene expression and protein synthesis involved in neuronal synaptic plasticity.38 Ketamine administration was restricted to the first 3 days of the combined treatment to explore whether short-term modulation of NMDA receptor signaling could affect analgesia induced by tramadol alone in the subsequent study days. Throughout the study period, any medication or treatment that the horses were currently receiving was continued.
Physiologic measurements—Systemic (systolic, mean, and diastolic) arterial blood pressures and heart and respiratory rates were assessed and intestinal sounds were auscultated before any drug administration (day 0 [baseline]) and then once daily for a period of 10 days (7 days during treatment and 3 days after treatment). These assessments were always performed at the same time of the day (between 2:00 pm and 3:00 pm) and by the same individual (DMH). Arterial blood pressures and heart rate were measured noninvasively with the horse in standing position with an oscillometric techniquef and the cuff placed at the base of the horse's tail. This method has been previously validated and used for measuring blood pressure in horses.39,40 The cuff width was equal to 40% of the circumference of the base of the tail. For each horse, blood pressure values were corrected for the difference in height between the heart (at the level of the scapulohumeral articulation) and the base of the tail; 0.75 mm Hg was added to the measured blood pressure value for each 1-cm difference. Three consecutive measurements were performed at each assessment; the mean was calculated and recorded for subsequent analysis. Respiratory rates were determined by observing thoracic excursions for a period of 1 minute. Intestinal sounds were auscultated and characterized by means of an index of gastrointestinal motility as previously described.41 Briefly, the upper and lower abdominal quadrants on the left and right side were auscultated; 2 sites within each quadrant were auscultated (duration of auscultation, 2 min/site). A subjective gastrointestinal motility score was assigned to each quadrant (0 = no bowel sounds; 1 = mild, low-pitched crepitation-like sounds that are audible 1 time/min at both sites within a quadrant; 2 = low-pitched crepitation-like sounds that are audible > 1 time/min at both sites within a quadrant; 3 = long and loud gurgling sounds that are audible 1 time/min at both sites within a quadrant; and 4 = long and loud gurgling sounds that are audible > 1 time/min at both sites within a quadrant). The scores were added for all 4 quadrants; the possible range of scores was 0 (no motility) to 16 (maximum motility). A gastrointestinal motility score ≥ 12 was considered normal and indicative of normal intestinal sounds and function.
Measurement of forelimb off-loading frequency and total forelimb load—Force plate analyses, as described previously,35 were performed to determine forelimb off-loading frequency and changes in total forelimb load bilaterally in horses before drug administration (day 0 [baseline]) and then once daily for a period of 10 days (7 days during treatment and 3 days after treatment). These assessments were always performed at the same time of the day (2:00 pm) and by the same individual (DMH). Briefly, a dedicated computerized system that was connected to 4 independent force plates quantified the mean load placed on each forelimb during a 5-minute period at a data sampling rate of 50 Hz. Each rectangular force plate, capable of measuring forces associated with weights of 0 to 500 kg, was calibrated (from 0 to 200 kg) regularly by task-specific software. The force plate signals were captured and digitized with a computer-based measurement system,g and the data were analyzed offline. The data collection process was automated, but the operator was able to pause data collection to allow repositioning of the digits on the plate if needed. All horses were trained to stand calmly in the measurement stanchion for at least 5 minutes and were loosely cross-tied to restrict movement of their heads and necks. No tranquilization or other forms of chemical restraint were used. Forelimb offloading frequency was defined as weight withdrawal from a specific limb that was > 1.5 SDs of the mean load of that limb, captured during the 5-minute measurement period and subsequently normalized per minute.
Blood sample collection and assessments of plasma TNF-α and TXB2 concentrations—A sample of jugular venous blood (10 mL) was collected in EDTA-containing tubes before (day 0 [baseline]), during, and after drug treatments for assessment of plasma concentrations of TNF-α and TXB2 (stable breakdown product of TXA2). Blood samples were centrifuged immediately after collection, and the plasma was harvested and frozen at −80°C until assayed. Plasma TNF-α concentration was measured once daily (days 0 through 10) in duplicate with an equine-specific ELISAh according to the manufacturer's recommendations. A mean concentration was calculated for each time point. Plasma TXB2 concentration was determined once daily (days 0, 1,3, 5, 7, and 10) in triplicate with a commercially available enzyme immunoassay kiti according to the manufacturer's guidelines. A mean was calculated to provide a value for each time point. Because there can be wide variation in plasma prostanoid concentrations among individual horses, the data were examined by calculating the percentage change from baseline for each horse, as performed in other studies.42,43
Behavioral responses—Horses were closely observed at least hourly throughout the study (10-day experimental period), and abnormal behaviors such as signs of depression or neuroexcitation were recorded. Drug administrations were to be discontinued and the horse removed from the study in the event of clinically important adverse behavioral responses.
Statistical analysis—Data were summarized and are expressed as mean ± SEM. Statistical analyses were performed with commercially available software.j Assessments were made of changes in each variable from baseline within a treatment as well as differences between treatments at each time point. Parametric data were analyzed with a 2-way repeated-measures ANOVA or t tests as appropriate, followed by Bonferroni posttests when significant differences between means were detected. Nonparametric data were subjected to the Kruskal-Wallis 1-way ANOVA on ranks and the post hoc Bonferroni test. Values of P < 0.05 were considered significant.
Results
Horses—The study horses included 8 mares and 7 geldings. Mean ± SEM age of the horses was 12.7 ± 1.3 years (range, 4 to 20 years), and mean weight was 460.2 ± 12.6 kg (range, 391 to 540 kg). Five breeds were represented: Arabian (n = 6), Quarter Horse (5), Paint Horse (2), Hanoverian (1), and Peruvian Paso (1). All 15 horses had laminitis of both forelimbs (duration, > 12 months). All horses had evidence of reduced total forelimb load before receiving treatment with tramadol alone or treatment with tramadol and ketamine; the mean ± SD total forelimb load profile was 52.3 ± 1.1% and 49.4 ± 1.8% of body weight, respectively. The normal load distribution of healthy horses is 58% of body weight. Only 1 horse was receiving NSAID treatment (phenylbutazone [2.5 mg/kg, PO, q 12 h]). That horse's laminitic condition did not change markedly during the experimental period, and the data from the horse were included in the final analysis.
Physiologic variables—On day 0, baseline values of heart rate, respiratory rate, and gastrointestinal motility scores were within reference limits for horses, whereas systemic blood pressure measurement revealed the presence of hypertension (mean systolic arterial blood pressure, 142.4 ± 7.2 mm Hg and 155.8 ± 8.6 mm Hg before treatment with tramadol alone and before treatment with tramadol and ketamine, respectively). Compared with baseline values, oral administration of tramadol alone every 12 hours for 7 days did not affect heart or respiratory rate (Figure 1) and gastrointestinal motility score (data not shown) during or after treatment. Treatment with tramadol alone resulted in a slight, nonsignificant decrease in systolic, mean, and diastolic arterial blood pressures (data for systolic and diastolic arterial blood pressures not shown), and the values were still indicative of mild hypertension. Similarly, the addition of ketamine during the initial 3 days of tramadol treatment did not change respiratory rate, heart rate, and gastrointestinal motility. However, significantly greater reductions in the systolic, mean, and diastolic arterial blood pressure values were associated with the combination treatment. The values were indicative of normotension after the fourth day. The blood pressure effects persisted during the combination treatment experiment, including the 3-day period following discontinuation of tramadol administration. Comparisons of daily data for the 2 groups revealed no differences in any of the physiologic variables at any time point before, during, and after either treatment.
Mean ± SEM respiratory rate (A and D), heart rate (B and E), and mean arterial blood pressure (C and F) in 15 horses with chronic laminitis of both forelimbs before (day 0 [baseline]), during (days 1 through 7), and after (days 8 through 10) oral administration of tramadol hydrochloride alone (5 mg/kg, PO, q 12 h [A, B, and C]) or oral administration of tramadol hydrochloride (5 mg/kg, PO, q 12 h) with IV administration of ketamine hydrochloride (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment [D, E, and F]). Horses received both treatments in a randomized crossover study (interval between treatments ≥ 60 days). *Bracketed values are significantly (P < 0.05) different from the baseline value. NS = Not significant (ie, bracketed values are not significantly [P ≥ 0.05] different from the baseline value).
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Forelimb off-loading frequency and total forelimb load—The off-loading frequency of each forelimb was quantified separately with the use of independent force plates, and the changes in mean forelimb load over time were measured as indicators of analgesia. Although all horses had laminitis in both forelimbs, one limb was usually more severely affected than the other (Figure 2). This limb difference was significant during the tramadol-ketamine treatment. Because of this nonuniformity, the mean off-loading frequency was calculated separately for the more lame and less lame forelimbs, and then a mean value was calculated for both forelimbs combined at each time point during the 2 treatments. For the more lame limb, tramadol alone reduced forelimb off-loading frequency during the first 3 days of treatment, compared with baseline findings; the off-loading frequency returned to the baseline value thereafter (Figure 3). No significant changes in the off-loading frequency were detected for the less lame limb; although the mean offloading frequency changed, those changes were not significant. Administration of ketamine at a subanesthetic dose during the first 3 days of tramadol treatment resulted in a significant reduction in the off-loading frequency of the more lame limb and in the mean off-loading of both forelimbs for the entire 7 days of drug treatment, although it was most pronounced during the first and second days of treatment (Figure 4). No changes were evident in the less lame limb, except at day 8 when the off-loading frequency was greater than the baseline value.
Mean ± SEM off-loading frequencies (indicative of the severity of lameness) for the more lame (squares) and less lame (circles) forelimbs in 15 horses with chronic laminitis of both fore-limbs before (day 0 [baseline]), during (days 1 through 7), and after (days 8 through 10) oral administration of tramadol alone (5 mg/kg, PO, q 12 h [A]) or oral administration of tramadol (5 mg/kg, PO, q 12 h) with IV administration of ketamine (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment [B]). Horses received both treatments in a randomized crossover study (interval between treatments ≥ 60 days). *At this time point, values are significantly (P < 0.05) different between forelimbs. NS = Not significant (ie, bracketed values are not significantly [P ≥ 0.05] different between forelimbs).
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Mean ± SEM off-loading frequencies for the more lame forelimb (A) and the less lame forelimb (B) and the mean off-loading frequency of both forelimbs (C) in 15 horses with chronic laminitis of both forelimbs before (day 0 [baseline]), during (days 1 through 7), and after (days 8 through 10) oral administration of tramadol alone (5 mg/kg, PO, q 12 h). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Mean ± SEM off-loading frequencies for the more lame forelimb (A) and the less lame forelimb (B) and the mean off-loading frequency of both forelimbs (C) in 15 horses with chronic laminitis of both forelimbs before (day 0 [baseline]), during (days 1 through 7), and after (days 8 through 10) oral administration of tramadol (5 mg/kg, PO, q 12 h) with IV administration of ketamine (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
When horses received tramadol alone, force plate measurements during the 3 days after cessation of treatment indicated that the off-loading frequencies of both forelimbs were similar to baseline values (Figure 3). In contrast (and in keeping with the pattern of blood pressure results), when horses received the tramadol-ketamine treatment, the off-loading frequency of the more lame limb was significantly lower than the baseline value (Figure 4). The mean off-loading frequencies for both forelimbs were also significantly lower than the baseline value on days 1 through 7 and on days 9 and 10. On day 8 (the first day after cessation of tramadol administration), the mean off-loading frequency was not different from the baseline value; however, the off-loading frequency of the less lame limb was significantly greater than the baseline value.
Daily changes (from baseline) in total forelimb load as a percentage of body weight were calculated as an additional estimate of pain relief. Results indicated that changes in total forelimb load from the respective baseline value were not significant throughout treatment with tramadol alone (days 1 to 7), although a significant change was evident following discontinuation of treatment (ie, on days 8 and 9; Figure 5). A larger significant increase from baseline was observed during and after tramadol-ketamine treatment, with the exception of findings on day 7.
Mean ± SEM changes (from the day 0 [baseline] value) in total forelimb load as a percentage of body weight in 15 horses with chronic laminitis of both forelimbs during (days 1 through 7) and after (days 8 through 10) oral administration of tramadol alone (5 mg/kg, PO, q 12 h [A]) or oral administration of tramadol (5 mg/kg, PO, q 12 h) with IV administration of ketamine (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment [B]). Horses received both treatments in a randomized crossover study (interval between treatments ≥ 60 days). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Plasma concentrations of TNF-α and TXB2—Baseline plasma concentrations of TNF-α were 1,208 ±575 pg/mL (range, 284 to 2,263 pg/mL) before administration of tramadol alone and 1,417 ± 334 pg/mL (range, 612 to 2,073 pg/mL) before administration of tramadol and ketamine (Figure 6). The values for the 2 groups were not significantly different. Compared with the respective baseline values, the reductions in plasma TNF-α concentrations on days 1 through 10 of treatment with tramadol alone were not significant; however, significant reductions were detected on days 4, 5, 6, and 7 of treatment with tramadol and ketamine.
Mean ± SEM plasma TNF-α concentration in 15 horses with chronic laminitis of both forelimbs before (day 0 [baseline]), during (days 1 through 7), and after (days 8 through 10) oral administration of tramadol alone (5 mg/kg, PO, q 12 h [A]) or oral administration of tramadol (5 mg/kg, PO, q 12 h) with IV administration of ketamine (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment [B]). Horses received both treatments in a randomized crossover study (interval between treatments ≥ 60 days). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Baseline plasma concentrations of TXB2 (a stable metabolite of TXA2) were 31.5 ± 10.0 pg/mL (range, 5.3 to 599.4 pg/mL) before administration of tramadol alone and 24.0 ± 3.4 pg/mL (range, 6.2 to 138.5 pg/mL) before administration of tramadol and ketamine. The values for the 2 groups were not significantly different. Compared with the baseline value, plasma TXB2 concentration remained unchanged during and after treatment with tramadol alone (Figure 7). Administration of tramadol and ketamine resulted in significant reductions in plasma TXB2 concentration from the baseline value on days 3,5, and 7 of treatment and on day 10 after cessation of treatment.
Mean ± SEM changes in plasma TXB2 concentration (stable metabolite of TXA2) in 15 horses with chronic laminitis of both forelimbs before (day 0 [baseline]), during (days 1, 3, 5, and 7), and after (day 10 [drug treatment was discontinued on day 8]) oral administration of tramadol alone (5 mg/kg, PO, q 12 h [A]) or oral administration of tramadol (5 mg/kg, PO, q 12 h) with IV administration of ketamine (0.6 mg/kg/h for 6 h/d for the first 3 days of tramadol treatment [B]). Horses received both treatments in a randomized crossover study (interval between treatments ≥ 60 days). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.610
Behavioral responses—No overt signs of excitation were evident when tramadol was administered alone. Neuroexcitation characterized by a heightened response to noise (3/15 horses) and muscle fasciculation (1/15 horses) developed during the ketamine infusions, whereas some horses (2/15 horses) appeared mildly sedated.
Discussion
The present study investigated the analgesic effects of pharmacological modulation of opioid, monoaminergic, and NMDA receptor systems by tramadol and ketamine treatment in horses with chronic laminitis of both forelimbs. Given the randomized, crossover design of the study, each horse served as its own control. Response to treatment was assessed by quantifying changes in forelimb off-loading frequencies and in total forelimb load, which are affected during chronic laminitis.4,35 Response to treatment was further assessed by monitoring changes in systemic arterial blood pressure variables and plasma concentrations of TNF-α (an important proinflammatory cytokine and mediator of neuropathic pain5,7–9) and TXB2 (a stable metabolite of TXA2, which is a vasoconstrictor prostanoid thought to be involved in the pathogenesis of laminitis44). The results indicated that tramadol alone provided limited pain relief and that the analgesia associated with tramadol administration could be significantly enhanced by coadministration of short-term infusions of subanesthetic doses of ketamine. This finding suggests that NMDA receptor–dependent mechanisms play a critical role in chronic laminitis-associated pain. There was evidence of modulation of cytokine and prostanoid production that might constitute, at least in part, additional underlying mechanisms for the analgesic and blood pressure effects observed in horses of the present study.
Elevated systemic arterial blood pressure in a population of ponies predisposed to laminitis has been reported,40 and similar findings were obtained before treatment in the chronically laminitic horses of the present study. Blood pressure variables remained elevated during administration of tramadol alone but decreased to normotensive values when ketamine was added to the treatment protocol. Interestingly, the reductions in blood pressure variables from baseline values in the present study were significant after the 3-day period during which ketamine was administered. In 1 investigation36 in healthy horses, ketamine infusions (delivered at a dose similar to that used in the present study) were associated with significant reductions in heart rate and mean arterial blood pressure 6 hours after the end of the infusion. Conversely, another study37 that used a higher dose revealed slight, nonsignificant elevations in heart rate and systemic blood pressure. These results suggest that blood pressure variables may have decreased during the hours following discontinuation of ketamine administration on each of the 3 days in the present study, but that such decreases were not detected because of the study design, or suggest that the typical cardiovascular stimulant effects of ketamine24 possibly prevented significant decreases in blood pressure variables in the hypertensive study horses. Respiratory and heart rates did not change significantly with either treatment in the horses of the present study. However, it has been reported that in healthy horses, respiratory rate increases and heart rate does not change during a cumulative IV infusion of tramadol.45 In another study,19 tachycardia was detected in healthy horses administered an IV bolus of tramadol (5 mg/kg); heart rate did not change when the same dose was administered orally. Respiratory and heart rates did not change in healthy horses following epidural administration of tramadol.46 These data suggest that the effects of tramadol on heart and respiratory rates appear to be related to the route, and possibly the rate, of drug administration. In healthy horses, infusions of ketamine at a dose similar to that used in the present study did not change respiratory rate,36 although that rate was reported to increase with higher doses.37
In the present study, tramadol alone reduced fore-limb off-loading frequency, compared with the baseline frequency, only during the first 3 days of treatment, indicating that the opioid and monoaminergic receptor effects of this drug are not sufficient for long-term nociceptive control in horses with chronic laminitis. Laminitis-associated pain stems from inflammatory and mechanical injury to the laminar tissue combined with neuropathic changes in the peripheral nervous system and CNS.1,4,35 Changes in nociceptive pathways associated with chronic laminitis likely lead to central sensitization and increased responses of nociceptive sensory neurons to both noxious and innocuous input.4 Thus, any reinjury of laminar tissue may lead to an increase in the severity of pain and subsequent breakthrough pain. In the present study, reinjury may have occurred secondary to the daily walk to and standing on the force plates, which are hard surfaces, or may have been associated with an increase in the use of the forelimbs during the initial period of pain relief. As such, it is possible that increases in pain severity may have developed as the study progressed and may explain the limited analgesia achieved via administration of tramadol. Alternatively, the bioavailability of tramadol may have decreased to subtherapeutic concentrations after 3 days of repetitive administration. We chose to administer tramadol at a dose of 5 mg/kg because bioavailability is good in horses at that dose,19 although some disagreement exists regarding the bioavailability of orally administered tramadol in equids,23 and on the basis of results obtained for the horse of the pilot study. Regardless, it appears that tramadol is unlikely to provide sustained pain relief in horses with chronic laminitis when administered alone at the dosage and route used in the present study.
Modulation of NMDA receptor activity by coadministration of subanesthetic doses of ketamine IV along with oral administration of tramadol resulted in markedly superior analgesia, compared with that achieved via oral administration of tramadol alone. It has been demonstrated that peripheral inflammation upregulates NMDA receptor expression in the spinal cord dorsal horn31 and switches the receptor voltage dependence toward a hyperpolarized state that can lead to hyperalgesia and allodynia.47 Ketamine-induced NMDA channel blockade is voltage dependent, with greater potency at hyperpolarized conditions, and also appears to be enhanced by agonist-induced receptor activation.48 Thus, changes in NMDA receptor function during peripheral inflammation should favor an increased sensitivity to ketamine, which substantiates the finding of the present study that superior analgesia resulted when subanesthetic doses of ketamine were added to the tramadol treatment. The results of the present study are further supported by the findings that ketamine and other NMDA receptor antagonists effectively reduce neuronal hyperexcitability and hyperalgesia associated with neuropathic pain27,49 and that considerable neuropathic changes have been identified in horses with laminitis.4 It is possible that the analgesia observed in horses undergoing tramadol-ketamine treatment could be exclusively due to ketamine, considering that tramadol alone had a limited analgesic effect. However, this is unlikely because the plasma clearance of ketamine is rapid in horses36,37 and the analgesia persisted for days after its discontinuation. Most likely, modulation of NMDA receptor–dependent changes in nociceptive pathways by ketamine27,34,38,47,49 was sufficient to potentiate or unmask the analgesic effects of tramadol. It is recognized that simultaneous pharmacological modulation of opioid and NMDA receptor signaling systems results in superior analgesia, compared with results when each system is modulated alone.34
Plasma TNF-α concentrations in the horses of the present study were approximately 4- to 5-fold higher than values (determined with the same assay technique) for healthy horses, yet similar to values for chronically laminitic horses of a previous study.10 There is abundant evidence that TNF-α, a pleiotropic proinflammatory cytokine, is upregulated in several conditions that appear clinically, at least in part, as painful neuropathies,18,50–52 including chronic laminitis in horses.10 The cytokine is produced and released following tissue injury by endoneural-associated cells, including Schwann cells, macrophages, endothelial cells, and fibroblasts.53,54 Exposure of peripheral nerves to TNF-α causes ectopic electrophysiological activity in nociceptive neurons,9 which has a role in the generation of hyperalgesia and inflammation.5 Tramadol alone did not significantly alter plasma TNF-α concentrations in the horses of the present study. To our knowledge, the effects of tramadol on plasma concentrations of TNF-α in horses has not been previously investigated; however, in humans who have neuropathic pain, tramadol reduces plasma concentrations of the cytokine.18 Significant reductions in the plasma TNF-α concentrations in parallel with improved analgesia were detected only when ketamine was added to the tramadol treatment protocol. This was somewhat expected because ketamine suppressed lipopolysaccharide-induced TNF-α secretion by an equine macrophage cell line in a dose-dependent manner.55 These findings indicate that important cross talk between NMDA receptor and TNF-α signaling systems likely exists and may be involved in the nociceptive mechanisms in horses with chronic laminitis.
In the horses of the present study, changes in blood pressure variables associated with the institution of analgesic treatment occurred in parallel with changes in plasma TXB2 (stable metabolite of TXA2) concentration, suggesting a possible role for TXA2 in the hypertension identified during baseline conditions. The baseline TXB2 concentrations in the horses of the present study were approximately 30-fold higher than values (determined with the same detection assay) in healthy horses.44 However, a direct causal relationship is difficult to ascertain because increases in plasma TX concentrations are not associated with increases in blood pressure variables in horses that are not affected by painful conditions.56 As such, in the horses of the present study, the reductions in blood pressure were likely secondary to decreased sympathetic activity because of pain relief and were not directly associated with circulating TX. Interestingly, TX is believed to contribute to the pathogenesis of laminitis by causing digital vasoconstriction and reducing blood flow.57 Data from endotoxemic mice have indicated that there is cross talk between TNF-α and TX receptor signaling pathways, which leads to microcirculatory dysfunction.58 These findings and results of the present study support further investigations of the use of ketamine and tramadol during the prodromal phases of laminitis not only for analgesia, but also for potential modification of pathogenesis of the disease.
The adverse effects observed in horses of the present study were of minor importance. Neuroexcitation associated with IV administration of ketamine36 and IV administration of tramadol19,23,45 in horses has been reported. In the present study, slight neuroexcitatory behaviors were observed in a few horses only during ketamine administration and did not require any intervention. Oral administration of tramadol alone did not induce any appreciable signs of neuroexcitation, as reported previously.19 Opioid receptor–mediated reduction in gastrointestinal motility is known to occur in horses and other species and is a major concern because it can result in ileus and colic, which can be fatal in horses. Transient and inconsequential decreases in intestinal sounds after IV administration of tramadol have been reported,45 but reductions in gastrointestinal sounds were not detected and colic did not develop in any of the horses of the present study. Tramadol has few serious gastrointestinal adverse effects in humans21 and appears to have a similar gastrointestinal adverse effect profile in horses when the drug is administered at the dosage and route used in the present study.
The present study has several limitations that should be considered. First, the precipitating factors that culminated in chronic laminitis as well as the precise duration of the condition were not known for most horses. However, all horses had chronic laminitis for > 1 year. We chose to study these horses instead of inducing the condition in healthy horses for humane and ethical reasons. Another limitation was that even though all horses had established and fairly stable laminitis, the condition did vary to some degree for each horse during the study. A randomized, crossover design was used with the intent of mitigating some of these effects. Upon inspection of the data, it was readily noticeable that the condition appeared to be less severe, as judged from the offloading frequency, total forelimb load, blood pressure variables, and plasma cytokine and prostanoid concentrations, prior to when the horses were to receive tramadol alone. The reason for this is uncertain, but might be related to modulation of NMDA receptor activity by ketamine. Ketamine can have long-term effects on several measures of chronic pain in humans32; however, to account for possible similar long-term effects in horses, there was a washout period of at least 2 months between treatments in the present study. Nevertheless, results of the study reported here strongly suggest that the NMDA receptor system has a pivotal role in chronic laminitis-associated pain. Lastly, pain associated with other structures, such as joints, muscles, and tendons, that was induced by unnatural postural changes was also likely present in the study horses. This would most certainly pose additional challenges for pain assessment with subjective scoring systems, but the use of force plates, which are very sensitive to changes in limb load, yields objective and reproducible measurements.35
Results of the present study indicated that a short-term infusion of ketamine at a subanesthetic dose in combination with oral administration of tramadol has immediate and lasting effects on several variables in horses with chronic laminitis-associated pain. These effects included a reduction in blood pressure variables and improvement in off-loading frequency and total load of the affected limbs, along with decreases in the plasma concentrations of TNF-α and TXA2 (indicated by measurements of TXB2). However, despite the improvement in lameness, many of the variables remained substantially elevated, compared with the values reported for healthy horses. Further studies aimed at understanding the mechanisms of this painful condition and testing of various treatment protocols are clearly warranted and much needed.
ABBREVIATIONS
| NMDA | N-methyl-d-aspartate |
| TNF | Tumor necrosis factor |
| TX | Thromboxane |
Tramadol hydrochloride 50-mg tablets, Amneal Pharmaceuticals LLC, Paterson, NJ.
Ketaset, Fort Dodge Animal Health Inc, Iowa.
Karo syrup, ACH Food Co Inc, Cordova, Tenn.
Angiocath, Becton, Dickinson & Co, Sandy, Utah.
AJ 5805 Portable PCA Syringe Pump, Angel Canada Enterprises Ltd, Burnaby, BC, Canada.
Cardell Model 9401 BP Monitor, Sharn Veterinary Inc, Tampa, Fla.
DATAQ Instruments Inc, Akron, Ohio.
TNF-α ELISA, Endogen Inc, Rockford, Ill.
Cayman Chemical Co, Ann Arbor, Mich.
GraphPad Prism, version 5.0c for MAC OS, GraphPad Software Inc, San Diego, Calif.
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