Tiletamine-Zolazepam for Total Intravenous Anesthesia in Sheep

Introduction

Sheep are an increasingly popular and convenient large animal model for biomedical research. General anesthesia is often required for prolonged invasive or noninvasive procedures. While inhalant anesthetics (eg, isoflurane) are commonly used anesthetics, partial or total intravenous anesthesia (PIVA or TIVA) with injectable anesthetics such as propofol, ketamine, or xylazine is also used.^1,2 While inhalant anesthesia provides reliable anesthesia and is easily titrated, it requires expensive equipment that may be incompatible with magnetic resonance imaging (MRI). The advantages of intravenous anesthesia as compared with inhalant include rapid onset of action, lower cost, and reduced environmental pollution and human safety hazards of exposure to volatile gases; the disadvantages include potential initial cost of the infusion pump, the cumulative effect of the PIVA/TIVA drugs that may slow recovery, and challenges in titrating the constituent medications.^3–6 Developing anesthetic protocols should focus on minimizing side effects and associated complications and a smooth yet quick recovery, while also considering cost, technical difficulty, analgesia level, and the impact on the procedure or study.

Many PIVA or TIVA protocols use a multimodal approach with a-2-adrenergic agonists, opioids, dissociatives, benzodiazepines, other injectable anesthetics, and local anesthetics.^1,4,7–13 A common ruminant TIVA field protocol includes xylazine, guaifenesin, and ketamine mixed in 5% dextrose in water, colloquially known as “ruminant triple drip.”^2,3,14–16 Constant rate infusions (CRIs) used in sheep include diazepam-ketamine,¹² detomidine,⁴ and propofol¹⁷ among others^{11,13–15,18,19} Alternatively, repeated bolus administration of intravenous anesthetics such as ketamine-diazepam, ketamine-xylazine, or ketamine-xylazine-guaifenesin is reported for TIVA, although this process can result in poor maintenance due to intermittent drug administration and increased risk of negative side effects, including hypoxemia, respiratory compromise, and cardiovascular depression.^3,5,20

Tiletamine, a potent nonnarcotic dissociative anesthetic agent related to ketamine, induces immobilization and anesthesia. Zolazepam is a benzodiazepine tranquilizer that induces muscle relaxation and has profound central nervous system effects.^21,22 Tiletamine-zolazepam (TZ) is a commercially available combination product widely used in laboratory animal, wildlife, zoologic, large- and small-animal medicine. It is frequently administered intramuscularly as an anesthetic premedication in the laboratory setting.^{2,3,8,16,17,20–26} TZ has recently been used as a CRI in dogs^5,10,27 with excellent outcomes, and for long-term deep sedation in an orangutan.²⁸ The benefits of TZ include reduced muscle rigidity, seizure-like activity, and a reduced injection volume compared with ketamine combinations when given at appropriate dosages, as well as a longer duration of action for many species.¹⁹ Using TZ may result in prolonged or turbulent recovery (eg, agitation, vocalization, myoclonic or convulsive activity, vomiting, or rapid excitability) due to species differences in metabolizing the 2 drugs.^2,22

Although TZ is used widely in laboratory animal medicine as an injectable sedative, to the authors’ knowledge, its safety and efficacy as a TIVA have not been studied in the large-animal laboratory setting. The objectives of this study were to evaluate the efficacy and clinical effects of the TZ TIVA protocol using a CRI in Dorset sheep undergoing a noninvasive imaging procedure.

Materials and Methods

Results

Direct arterial blood pressure levels (SAP, DAP, and MAP) were stable throughout the procedure, with no statistically significant differences at any time point. Overall mean SAP was 104.4 ± 1.3 mm Hg (P = 0.42), DAP was 80.9 ± 1.1 mm Hg (P = 0.38), and MAP was 91.5 ± 1.2 mm Hg (P = 0.32) (Figure 1).

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HR did not differ at any time point as compared with all other time points (P = 0.71). Mean HR was 112 ± 9.88 bpm at T₀ and 99.67 ± 2.69 bpm at time 120 (T₁₂₀; Figure 2). The average HR throughout the procedure was 104.9 ± 1.7 bpm.

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Similarly, body temperature remained stable and with no statistically significant changes throughout the procedure (P = 0.24). Mean body temperature was 101.51 ± 0.04 °F throughout the procedure (Figure 3). ETCO₂ also remained stable throughout with no statistically significant differences at individual time points (P = 0.23). Mean ETCO₂ was 36.87 ± 0.43 mm Hg throughout the procedure.

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aBG analysis showed that HCO₃ and pH decreased from baseline to the end of the procedure, while pCO₂ and BG levels increased slightly, but the changes were not statistically significant (Table 1).

All sheep recovered uneventfully with no reported signs of emergence delirium or turbulent recovery. Mean time to extubation was 25 ± 6.5 minutes, while mean time to standing recovery was 34 ± 8.0 minutes after discontinuation of the TZ CRI. While the length of anesthesia for each individual varied, each animal was solely maintained on TZ CRI throughout the entirety of their procedure; therefore, time to extubation and recovery from anesthesia was assessed as equivalent.

Discussion

This study demonstrates that TZ CRI is an effective TIVA method in sheep and provides a stable anesthetic plane for noninvasive imaging procedures. Our findings show that (1) HR, ETCO₂, body temperature, SAP, MAP, and DAP did not differ significantly at any time point; (2) recovery was smooth with no differences in time to extubation or recovery between individuals; and (3) aBG was not significantly impacted by the TZ CRI over time.

To the authors’ knowledge, this is the first study evaluating the efficacy and utility of TZ as a CRI for noninvasive imaging procedures in sheep. TZ is used frequently in species such as pigs, dogs, primates, and other zoo or wildlife species for sedation and anesthesia induction as a single intramuscular or intravenous injection,^7,21,22,31 and this study sought to further investigate TZ’s effectiveness and usefulness as a CRI in sheep. Doses were based on the clinical experience of the authors with several pilot studies (data not reported) determining that 10 mg/kg/h was an appropriate average starting CRI rate. The CRI was titrated to effect and adjusted throughout the procedure between 5 and 15 mg/kg/h as needed, with a majority of the individuals being stably maintained on 10-12 mg/kg/h without frequent adjustments to the CRI rate. Sheep were monitored by experienced laboratory animal veterinarians, and titrations were made at 2 mg/kg/h based on physiologic indications that the animal was not in a good plane of anesthesia (eg, HR, jaw tone, and voluntary movement). Because TZ was the sole anesthetic agent, we expected a higher required dose to prevent arousal and to counteract stimulation from noise in the MRI machine and manipulation related to the transcranial ultrasound. Previous studies found a CRI rate of 2 mg/kg/h provided appropriate and adequate TZ CRI anesthesia in dogs as both PIVA and TIVA.^5,10,27 Sedation and induction TZ doses in sheep have previously been reported between 12 and 24 mg/kg IV,^22,32 indicating sheep may require higher drug doses compared with companion animals.³³

In this study, HR, arterial blood pressure, and temperature were not significantly affected across all time points. Results from TZ CRI studies²⁷ in dogs showed a significant transient tachycardia after induction and a gradual but significant body temperature decrease. Dissociative anesthetics (such as tiletamine and ketamine) induce catecholamine release from the sympathetic nervous system and transient HR increases.¹⁹ This sympathomimetic action indirectly supports the cardiovascular system and may help maintain HR and blood pressure when used as a CRI as seen in this study. Statistically significant hypothermia was documented with TZ use in dogs undergoing surgical manipulation under anesthesia for 50-70 minutes^5,10,27 and in sheep for 90-120 minutes.^22,24 The sheep in this study were covered with towels or blankets while in the MRI machine to help prevent thermal loss, which may have prevented significant temperature fluctuations; the MRI’s confined space and the procedure’s noninvasive nature likely contributed to decreased heat loss. Because the sheep were shorn just prior to delivery to the facility, their wool was less than 1 inch thick and likely did not contribute greatly toward thermoregulation and heat trapping. It is unlikely that the noninvasive imaging performed (MRI and transcranial ultrasound) would affect any physiologic values or the anesthesia as a whole.

Previous studies where TZ was administered intramuscularly or intravenously provided 25-40 minutes of anesthesia in sheep,^2,22 which is consistent with the average time to exubation (25 minutes) and recovery (34 minutes) for this study. Anesthetic recovery differences across species have been documented and may be related to anesthetic drug metabolism rates. In species with a longer tiletamine duration of action (eg, dogs), emergence delirium is reported, likely related to the effects of dissociative anesthetics causing muscle stiffness and sympathetic stimulation.^5,32 The sheep in this study had smooth recoveries without emergence delirium or hyperactivity, suggesting zolazepam persists longer than tiletamine. Other investigators³ found that recovery from a continuous ruminant triple drip (50 mg xylazine, 500 mg ketamine, and 500 mL guaifenesin) at a 2.2-mg/kg/h infusion resulted in an average recovery time of over 98 minutes, which is significantly longer than the recovery time seen in our study. The same study attributed longer “triple drip” recovery times compared with xylazine-ketamine to the guaifenesin’s muscle relaxant effects and slower plasma drug elimination, which is likely why this study’s sheep had shorter recovery times.

The study we describe here had several limitations. The first limitation was the exclusive use of healthy, young male Dorset sheep. Individual age, sex, and breed-related effects may result in altered anesthesia outcomes.^34,35 The second limitation was that the study’s goal was to evaluate TZ TIVA efficacy during a noninvasive imaging procedure; therefore, the impact of painful or surgical stimuli was unassessed. Although ketamine reportedly provides somatic analgesia,³⁶ tiletamine or TZ is ineffective at providing somatic analgesia.^{5,13,22,37,38} However, sufficient analgesia may occur when TZ is combined with xylazine or butorphanol^{1,12,31,37,38} or when used as PIVA with inhaled anesthetics.^10,27 While our results support the finding that TZ CRI is appropriate for noninvasive procedures where a similar level of anesthesia is needed, this method may be inappropriate for invasive, surgical, or those procedures providing significant patient stimulation. Third, full bloodwork assessment (CBC and serum chemistry) was not performed in this study; therefore, clinical chemistry alterations during or after anesthesia were unassessed outside of the blood gas analytes. Patil et al²⁷ assessed hematologic and biochemical parameters in TZ CRI anesthetized dogs and found mild, transient, and nonsignificant changes in several parameters. Fourth, a small dosing range (5-15 mg/kg/h) was used for this study, with each animal being titrated to maintain appropriate anesthesia for the MRIs. This approach more accurately reflects real-life anesthetic use but does not allow for the study of a wider range or multiple anesthetic dose levels.

Mechanical ventilation was used in all study procedures, which affords better respiratory and circulatory control compared with spontaneously breathing anesthetized animals. In general, dissociative anesthetics cause an apneustic breathing pattern characterized by breath holding followed by several shallow breaths.^19,22 Studies³² have demonstrated that TZ produces dose-dependent respiratory depression in multiple species. In this study, the sheep were mechanically ventilated to maintain ETCO₂ between 35 and 55 mm Hg due to the nature of the imaging experiment, so apneustic breathing was not observed, and respiratory depression was not appreciated. Mechanical ventilation may have benefitted the sheep in this study by maintaining minute ventilation and tidal volume throughout the procedure.