Materials and Methods

Animals—Twelve healthy adult horses were used in 2 experiments. Six healthy adult horses (3 males and 3 females; mean ± SD body weight, 360 ± 24 kg) were used to evaluate SLA, and 6 other healthy adult horses (3 males and 3 females; body weight, 396 ± 30 kg) were used to evaluate antinociceptive effects of buprenorphine. Health status was assessed on the basis of results of clinical examination, a CBC, and blood gas analysis. The study was approved by an institutional animal care committee (protocol No. 107/2004).

Experimental design—Each horse received 3 treatments (10 mL of saline [0.9% NaCl] solution, 5 μg of buprenorphine^a/kg, and 10 μg of buprenorphine^a/kg). Treatments were administered IV. Order of treatments was randomized, and there was a 10-day interval between subsequent treatments. The day before an experiment, each horse was placed in a behavioral stall (16 X 16 m²) for habituation to the environment. During the first day in the behavioral stall, each horse was fed 2 kg of a pelleted commercial food^b and allowed ad libitum access to hay and water. Each horse was fed the pelleted feed and provided fresh hay the following morning (day of the experiment); horses were allowed to eat for 1 hour, and all remaining food was then removed from the stall.

Evaluation of SLA—Horses were not restrained when in the behavioral stall, and disturbances were kept to a minimum. The SLA was investigated in accordance with the method described in another study.¹⁵ The SLA was recorded by pulses generated from 8 juxtaposed photoelectric sensors^c that emitted an infrared beam. The photoelectric sensors were spaced equally around the stall at a height of 45 cm. A pulse was generated each time the beam was interrupted by the horse. The number of pulses per minute was counted; information was stored in a data recorder^d connected to a microcomputer for subsequent analysis.

The SLA was recorded for 30 minutes to establish baseline values. Saline solution, 5 μg of buprenorphine/kg, or 10 μg of buprenorphine/kg was then injected. Treatments were administered IV into a jugular vein during a period of 5 seconds. Completion of the injection was designated as time 0. The SLA was recorded at 5, 10, 15, 30, 60, 90, and 120 minutes and thereafter at 2-hour intervals for 16 hours after administration of the treatment. At those same time points, behavior was observed through a small window with the observer positioned outside the stall. Signs of excitement (restlessness, head nodding, digging, shifting of limbs, vocalizing, trotting, and galloping) were recorded but not quantified.

Antinociception evaluation—Conditions were the same with regard to habituation and feeding for horses as those described for the preceding experiment. Disturbances were kept to a minimum, and horses were restrained by use of a halter only during antinociception evaluation.

Response to nociception was evaluated with a heat projection lamp by use of methods adapted from other studies.^16,17 Painful stimuli were applied at 2 sites by rapid exposure to the heat lamp. Application at the first site (lateral surface of the proximal phalanx of the thoracic limb) was used to measure HWRL, which was defined as the amount of time elapsed between focusing the light beam and limb withdrawal. Application at the second site (dorsal point of the shoulders of the horse) was used to measure STRL, which was defined as the amount of time between focusing the light beam and detection of skin twitching.

Hair was shaved from the regions of stimulation, and black water-based ink was painted on the skin before application of the focused light. Use of the ink was intended to provide uniform light reflection and heat absorption. Each latency reflex was obtained by use of a precision timer^e with accuracy measured to 0.01 seconds. The timer was adapted to the equipment and turned on and off simultaneously with the heat lamp. Five repetitions were performed. The highest and lowest values were removed, and the mean of the remaining 3 values was used for data analysis. The painful stimulus was applied for a maximum of 10 seconds to ensure that the tissues were not injured. A secondary nonfocused lamp was substituted frequently to ensure that the horses did not develop a conditioned response to light perception rather than to the pain perception caused by the heat of the focused light beam.

Both HWRL and STRL were measured by the same observer (ABC) to establish baseline values. Thirty minutes later, each horse was administered 1 of the 3 treatments (saline solution, 5 μg of buprenorphine/kg, or 10 μg of buprenorphine/kg) as an IV injection. Order of treatments was randomized, and injections were administered into a jugular vein during a period of 5 seconds (time of completion of each injection was designated as time 0). Then, HWRL and STRL were again measured at 0, 5, 15, 30, 60, and 90 minutes and 2, 3, 4, 5, 6, 7, 8, and 11 hours after treatment.

Statistical analysis—Statistical analysis was performed in accordance with the method described elsewhere¹⁸ by use of commercial software.^f Results were expressed as mean ± SEM. Differences among groups at each time point were compared by use of an ANOVA followed by the Tukey test. Differences were considered significant at values of P < 0.05.

Results

Evaluation of SLA—Excitement was observed in all horses from 5 to 10 minutes after administration of both doses of buprenorphine. Characteristic findings of excitement were restlessness, continuous head nodding, digging, shifting of limbs, vocalizing, trotting, and even galloping.

The SLA was uniform in all horses by 30 minutes after the adaptation period. The light beam was interrupted approximately 10 times/min in all groups of horses before any treatments were administered (Figure 1).

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Mean ± SEM values for SLA measured by interruption of light beams emitted from photoelectric sensors after IV administration of saline (0.9% NaCl) solution (circles), 5 μg of buprenorphine/kg (inverted triangles), or 10 μg of buprenorphine/kg (triangles) to 6 horses. There was a 10-day interval between subsequent treatments. Completion of injections was designated as time 0. ^a,b,cWithin a time point, values with different letters differ significantly (P < 0.05; Tukey test).

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.246

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The SLA was significantly higher from 1 to 6 hours after administration of 5 μg of buprenorphine/kg and from 15 minutes to 14 hours after administration of 10 μg of buprenorphine/kg, compared with results after administration of saline solution (Figure 1). The SLA was higher from 30 minutes to 14 hours after administration of 10 μg of buprenorphine/kg, compared with the SLA after administration of 5 μg of buprenorphine/kg.

Antinociception evaluation—The HWRL was approximately 2 seconds in horses after administration of saline solution. The HWRL was significantly longer at 30 minutes after administration of 5 μg of buprenorphine/kg, compared with the HWRL after administration of saline solution (Figure 2). The HWRL was significantly longer at all time points after administration of 5 μg of buprenorphine/kg, compared with results after administration of 5 μg of buprenorphine/kg or saline solution.

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Mean ± SEM values for HWRL measured after IV administration of saline solution, 5 μg of buprenorphine/kg, or 10 μg of buprenorphine/kg to 6 horses. See Figure 1 for key.

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.246

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We did not detect a significant difference in STRL when horses were administered 5 μg of buprenorphine/kg or saline solution (Figure 3). The STRL was significantly longer between 1 and 6 hours after administration of 10 μg of buprenorphine/ kg (except at 2 and 4 hours after administration), compared with the STRL after administration of 5 μg of buprenorphine/kg or saline solution.

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Mean ± SEM values for STRL measured after IV administration of saline solution, 5 μg of buprenorphine/kg, or 10 μg of buprenorphine/kg to 6 horses. See Figure 1 for key.

Citation: American Journal of Veterinary Research 68, 3; 10.2460/ajvr.68.3.246

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Discussion

The IV administration of buprenorphine to horses resulted in a short latency period for increases in SLA as well as for antinociception. In cats, pressure and thermal thresholds increased only at 30 and 45 minutes, respectively, after SC administration of buprenorphine.¹⁹ The fact that the cats of that study were administered buprenorphine by the SC route of administration, whereas the horses of the study reported here were administered buprenorphine by the IV route of administration, could explain the reason we detected a shorter time until onset for the horses of our study.

Various routes of administration in cats can achieve similar plasma buprenorphine concentrations but yield differing maximum increases in thermal threshold above baseline values. Thermal threshold was approximately 12°C above baseline after IV injection of 20 μg of buprenorphine/kg, 10°C after mucosal administration of 20 μg of buprenorphine/kg, and 4°C after IM administration of 10 μg of buprenorphine/kg,^20,21 which revealed that the route of administration of buprenorphine is important for enabling it to reach the effector site. Routes that result in slow uptake may not achieve sufficient concentration gradients to drive the drug into the biophase.

The short time of onset for buprenorphine in horses may be related to the lipophilic characteristics of the molecule, which enable it to quickly bind to opioid receptors in the CNS. In the study reported here, SLA increased more rapidly and for a longer time after administration of 10 μg of buprenorphine/kg. This may have been associated with the high affinity and intrinsic activity of buprenorphine at opioid receptors, which caused prolonged and dose-dependent effects.

Other opioids, such as fentanyl and apomorphine, also increase SLA in horses. Although both of those opioids are pure agonists, their mechanism of action on SLA apparently differs.^3,4 Authors of those studies believe it is likely that fentanyl, but not apomorphine, acts directly on OP3 (μ) receptors because the effects are antagonized by naloxone. Apomorphine increases dopamine release in rats, even in those with lesions in the dopaminergic tract, which indicates that there is no need for a central pathway for the mechanism of action. This effect is comparable to the effect for anfetamine.

The increase in SLA after the administration of buprenorphine in horses may result from activation of dopaminergic pathways. Dopamine, a noradrenaline precursor, is the main catecholamine in the extrapyramidal system of mammals.⁷ The striatus body, which is the part of the extrapyramidal motor system related to movement, has a high concentration of dopamine.²² Catalepsia, stereotyped movements, and increased concentrations of homovalinic acid in the proencephalus have been reported⁸ in rodents administered buprenorphine; however, differences among species with regard to how buprenorphine exerts its effects should be considered, and additional studies will be necessary to provide evidence that supports the hypothesis that buprenorphine activates dopaminergic pathways in horses in the same manner that it does in rodents.

Opioid-induced excitement is widely described in healthy people. This excitement can even lead to convulsions.²³ The dose of an opioid necessary to induce excitement is lower in cats, horses, and humans than it is in dogs and monkeys.²⁴ Opioid-induced excitement has also been observed in healthy horses, even with the use of partial opioid receptor agonists or agonist-antagonist opioids.3,4,12,25,26 The risk of adverse effects after administration of a dose of morphine in horses is inversely proportional to the intensity of pain.²⁷ It appears, however, that excitement is minimized or may not be observed in horses that have clinical signs associated with pain and are treated by administration of opioids. This was confirmed in a retrospective study²⁸ of the postoperative use of morphine in horses in which investigators concluded that the use of morphine was not associated with a greater incidence of adverse effects and may be indicated for use in horses to control or alleviate pain.

In the study reported here, we detected a dose-dependent increase in SLA but analgesia was not observed with the lower dose of 5 μg of buprenorphine/kg. We anticipated that there would be a similar antinociceptive response between the STRL and HWRL; however, analgesia was more prolonged for the HWRL, compared with that for the STRL, after administration of 10 μg of buprenorphine/kg. The possible effect of SLA on the HWRL may explain differences observed in antino-ciception induced by buprenorphine. A possible error in nociceptive evaluation of the HWRL by use of a thermal stimulus at the phalanx was that an increase in SLA may decrease HWRL; however, in the study reported here, the opposite result was achieved.

To reduce the effects of SLA on HWRL, we used an alternative method adapted by one of the authors (AQ-N) from methods described in other studies.^17,29 For this method, the heat was projected to the dorsal point of the shoulders until cutaneous twitching was observed. According to authors of those other studies,^17,29 the STRL would be more appropriate than the HWRL for investigating the effects of sedatives and opioids because sedatives could delay the HWRL as a result of lethargy and opioids could decrease the HWRL as a result of an increase in SLA.

In the study reported here, the small antinociception observed with the STRL nociceptive test was probably biased by the increased muscular tone and twitching typically observed with the use of opioids in horses, which has been described in ponies treated with morphine or butorphanol²⁵ and horses treated with alfentanyl.²⁸ These findings suggest that even if the increase in SLA interfered with the HWRL, this method would appear to be better than the STRL for evaluating antinociception induced by administration of opioids to horses.

For the HWRL, the higher dose of buprenorphine in horses yielded similar antinociception, compared with other reports^30–33 on the use of buprenorphine in other species. In pigs, a decrease in the response to thermal stimulation lasted for 7 hours after administration of 10 μg of buprenorphine/kg and a dose of 5 μg/kg was virtually ineffective.³⁰ In sheep, administration of 6 μg of buprenorphine/kg induced analgesia for 3 hours (as measured by use of thermal stimulation), although there was no analgesic response evident for mechanical stimulation.³¹ Values for a visual analogue scale were reduced for 7 hours after administration of 10 μg of buprenorphine/kg to cats undergoing elective surgeries; in addition, the threshold to thermal stimulation was increased for 12 hours.^11,32 In dogs undergoing arthrothomy, administration of 10 μg of buprenorphine/kg induced analgesia for 7 hours.³³

Doses of < 10 μg of buprenorphine/kg are virtually ineffective in some species.^30,31,34 The same result was observed for HWRL in horses in the study reported here. In our study, administration of 10 μg of buprenorphine/kg induced long-lasting analgesia for 11 hours, which is consistent with data reported^{10,11,30,32,33} for other species.

Administration of buprenorphine induced a dosedependent increase in SLA, which should be considered a potential problem in healthy horses. Administration of a dose of 10 μg of buprenorphine/kg resulted in antinociception ranging up to 6 (STRL) or 11 hours (HWRL) after administration in horses. Administration of a dose of 5 μg of buprenorphine/kg did not yield this same antinociception by use of our experimental model.