Methods

This study was designed as a prospective, blinded, crossover experimental study. The protocol was approved by the University of Pennsylvania Institutional Animal and Use Committee (no. 806698). An a priori power analysis (α = 0.05, β = 0.8, effect size f = 3) revealed that a minimum of 6 horses would be necessary to detect significant differences in TTs (6.5 °C).

Instrumentation

The day before the experiment and after physical and lameness screening examinations, a 14 gauge, 13.3 cm catheter (MILA International Inc) was inserted in a jugular vein under aseptic conditions. The horses were then sedated using detomidine (0.01 mg/kg) IV and a caudal epidural catheter (Arrow® FlexBlockTM Continuous Peripheral Nerve Block Kit, Teleflex®) was inserted at the first intercoccygeal space. After insertion of the 17 Ga X 7.6 cm Tuohy needle into the epidural space and confirmation of the correct epidural location using the hanging drop and loss of resistance techniques, the catheter was advanced to a distance of approximately 8 cm. The free end of the catheter was connected with the provided syringe adapter and filter. The catheter was then secured to the skin using a 2-0 nylon suture, and a dressing was placed over the site for protection. Every 12 hours the catheter was flushed with 1 mL heparinized saline (0.9% NaCl) solution and assessed for increased resistance to injection.

Finally, a 3 X 3 cm site over the left and right withers (Location A), cranial (Location B), and caudal (Location C) abdominal areas, the area over the tuber coxae (Location D) as well as over left and right hind limb dorsal pastern regions (Location E) were clipped and shaved (Figure 1) in preparation for placement of the TT sensors. The location of the sensors in a horse fitted with the wireless TT testing device is shown in Figure 1. Horses were then returned to the stall for the day and night before the first day of the experiment.

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Position of the thermal probe on the horse during the measurements (modified from Söbbeler FJ, Kästner SB. Effects of transdermal lidocaine or lidocaine with prilocaine or tetracaine on mechanical superficial sensation and nociceptive thermal thresholds in horses. Vet Anaesth Analg. 2018;45(2):227–233. doi:10.1016/j.vaa.2017.10.003).

Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.07.0166

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Results

Throughout the experiment, there were no significant changes in skin temperature under each sensor observed across all treatment groups. The placement of the thermal probe on the skin did not cause any negative effects such as burns or redness at any point during the study. After the administration of intravenous detomidine, all horses became mildly to moderately sedated. Additionally, no adverse effects or behavioral changes were observed in any of the subjects after placement of the caudal epidural catheter and epidural injection of morphine and xylazine. The fecal output of the horses remained consistent over the course of 2 days regardless of the treatment received.

Results of TT testing at increasing dilutional volumes over all timepoints are shown (Table 1). No significant differences were found in the baseline TT across different locations. When the 25 mL epidural volume was administered, location E consistently reached the cut-out temperature for all measured timepoints, while other locations did not significantly differ from the baseline. With the administration of 35 mL TTs in location D was significantly higher than the baseline TT for up to 8 hours, and location E exhibited a significantly increased TT at all timepoints. At 50 mL dilution, TTs at all the timepoints at Locations D and E were significantly higher than the baseline temperature. At location C the TTs were higher than baseline at every timepoint, but statistical significance was observed only at 4, 6, and 8 hours. With a 75 mL volume TTs at locations E, D, and C were significantly increased from the baseline TT. At location B the TTs were higher than the baseline at every timepoint, but statistical significance was observed only at 2 and 10 hours. Lastly, when 100 mL of epidural volume was administered TTs at locations E, D, and C were all significantly higher than the baseline TT. At locations B and A the temperatures were higher than the baseline at every timepoint, but statistical significance was observed only at 4, 8, and 10 hours.

Discussion

This study confirms that the dilutional volume of epidurally administered morphine and xylazine, without altering the drug dose, impacts the cranial spread of regional analgesic effects in healthy standing horses as measured by TT testing. This is in agreement with previous data that show xylazine administered epidurally at a dose of 0.15 mg/kg and a volume of 0.15 mL/kg (that corresponds to the 75 mL dose in this study) was successful at decreasing the halothane MAC for ponies stimulated with an electrical shock over the palmar/plantar digital nerves of both the thoracic and pelvic limbs.¹⁵ Additionally, it has been demonstrated that 20–40 mL of methylene blue administered epidurally at the intercoccygeal level migrates across 12 vertebral spaces in horses suggesting that our volume of 100 mL should be sufficient to reach the withers.¹⁶ In horses, experimentally induced joint pain in the thoracic limbs was effectively controlled by epidural morphine diluted to a total volume of 0.15 mL/kg and injected at a rate of 10 s/mL.⁷ Other studies have recommended a dose of 0.2 mL/kg for effective analgesia at the thoracic limb.⁸ Our study revealed a significant increase in TTs at the withers compared to baseline only with the maximum volume of 100 mL (that corresponds to approximately 0.2 mL/kg) whereas a volume of 75 ml total (approximately 0.15 ml/kg) had no significant effect at that location. It is possible that with a higher dose of morphine, the TT at the withers may be affected at a 75 mL volume. Titrating both the dose and injectate volume should be explored using TT as a measure of nociception in the future. However, high epidural pressure can cause a rise in CSF pressure. Therefore, it has been suggested that the use of large-volume epidural injectates can induce hypotension and cardiovascular depression which, in horses, can result in ataxia, potentially dangerous for animals and handlers. ^17,18

It has been shown for epidurally administered morphine that a total volume of 5 mL was not sufficient to provide analgesia in the thoracic limb of anesthetized ponies undergoing orthopedic surgery of the forelimb.¹⁹ This suggests that the cranial migration of the small volume of drugs was insufficient to activate the opioid receptors in the cervical and thoracic regions of the spinal cord.^16,20 To further support the need for a higher injectate volume, a study in goats has shown that the cranial spread of a solution injected epidurally is directly related to the volume of injectate.⁶ It is important to remember that the concentration of a drug has been shown to influence the onset and quality of the block, and the dose of a drug is the product of its concentration and volume. One challenge of interpreting the findings of most studies aiming to define the influence of any one of these factors is that modifications in one variable automatically produce changes in another. Although the largest dilution volume allows for more cranial spread, the greater dilution further decreases the concentration of the drug.

Regarding the onset of analgesia, several studies in both horses and humans have shown that epidural administration of xylazine and morphine induces analgesia within 1 hour and provides long-lasting pain relief.^21,22 The findings of our study agree with this timeline as all treatments resulted in an increase in TT at the first testing time point after administration, apart from location A with volumes less than 100 mL. This difference in behavior at the most cranial location is an interesting finding of this study as the difference between baseline TT and the TT after morphine and xylazine administration was smaller (∼3 degrees) at the most cranial location (location A) compared to all other locations (∼5–7 degrees). This decrease in the difference between baseline TT and postepidural TT may indicate reduced analgesic efficacy. The reason why analgesic efficacy appears to be reduced at the most cranial locations is unknown and likely depends on multiple factors. After crossing the dura mater, lipid-soluble drugs tend to bind to the receptors in the spinal cord, while water-soluble drugs tend to remain in the CSF.²³ Opioid and α-2 adrenergic receptors are codistributed in the substantia gelatinosa layer of the dorsal horn of the spinal cord and analgesic effects are dependent on the concentration of ligand molecules binding to these receptors.²⁴ Therefore, effects are influenced by the molecules diluted in the CSF or absorbed in the spinal tissue, as well as the dissociation kinetics of the drug.²⁵ Consequently, it is possible these factors lead to a decrease in the relative number of available molecules binding to receptors in more cranial regions, resulting in reduced analgesia in dermatomes further from the injection site.

Highly lipid-soluble opioids such as fentanyl and alfentanil are rapidly absorbed from the epidural space to the subarachnoid space and plasma after epidural administration²⁴ and, therefore, cranial movement of lipid-soluble opioids can be limited by their uptake into the spinal cord after transfer into the subarachnoid space.¹⁶ In contrast, lesser lipid-soluble drugs such as morphine will tend to linger in the water phase of CSF and act on receptors in the substantia gelatinosa of the dorsal horn of the spinal cord, inhibiting the release of substance P.⁴ Spread by CSF bulk flow might be greater than with highly lipid-soluble drugs and cranial migration of epidural morphine might be independent on the diluent volume.^23,26 Xylazine, like morphine, is thought to be absorbed into the CSF and act on receptors in the substantia gelatinosa of the dorsal horn of the spinal cord. Both drugs act by inhibiting the release of substance P from the A δ and C fibers of the spinal cord and their 2 independent receptor-mediated mechanisms act synergistically to inhibit pain.²⁷ In horses, the specific location, density, and subtypes of spinal opioid and α-2 adrenergic receptors have not been described. Therefore, it is difficult to determine how each drug in the present study had its respective effects and whether morphine was potentiated by the presence of xylazine.

It appears that lipid and water solubility are important factors, but other considerations such as molecular shape and receptor binding may also affect transfer across dura, uptake into neural and perineural tissue, and dilution into the CSF.²⁴

To evaluate the analgesic effect of different epidural volumes, we used nociceptive thermal threshold testing with thermo-based contact heat. One advantage of a thermode based system is that it can be adjusted so that the increase in temperature is linear. Another advantage in horses is that it is possible to maintain a relatively constant skin contact pressure using a thermode, standardizing the rate of thermal transfer.²⁸

Our study has several limitations that need to be acknowledged. Firstly, a larger sample size could have been beneficial to assess the effects of the treatments; however, our a priori power analysis indicated that a sample size of 6 should result in robust results. Secondly, exploring higher volumes and different dosages could have been interesting to evaluate. This would have allowed for a more comprehensive understanding of the relationship between volume and analgesic outcomes. Additionally, the chosen interval of 3 days between treatments might not have been sufficient to fully evaluate the effects and potential carry-over of the previous treatments. Considering a longer interval between treatments would have allowed for a greater washout period. However, this short washout period allowed for the completion of this study using the same catheters left in place reducing the risk of malplacement. Further, as baseline testing was performed before every treatment it is not likely that the washout period interfered with the results we obtained.

The regional effects of caudal epidural analgesia in horses are influenced by the volume of the administered solution. This study showed that horses can safely be administered epidural injectate volumes up to 100 mL with 0.2 mg/kg morphine and xylazine and that a significantly increased TT is seen at the level of the withers. Clinically this is relevant as pain management of the thorax, abdomen, and forelimbs is a chronic problem in equine management. While more testing will be required to fully optimize this technique, this study indicates that with enough volume a caudal epidural catheter could play a role in pain control of these anatomic areas. However, it is important to note that as the treatment targets more cranial locations of pain with increased dilutional volume, the efficacy and degree of the analgesia may be reduced.

Disclosures

The study was approved by the University of Pennsylvania Institutional Animal and Use Committee (No. 806698).

No AI-assisted technologies were used in the generation of this manuscript.

Funding

Intramural funding was provided by the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania.