<I>Otolemur Garnettii</I

A Comparison of the Efficacy and Cardiopulmonary Effects of 3 Different Sedation Protocols in Otolemur garnettii

Kelsey R Finnie,1,* Carissa P Jones,1 William D Dupont,2 Kenneth J Salleng,1 and Katherine A Shuster1

The Northern greater galago (Otolemur garnettii) is a prosimian primate most commonly used to study the evolutionary development of vision and somatosensation. This study aimed to investigate the efficacy and cardiopulmonary effects of 3 sedation protocols commonly used in other primate species: 1) alfaxalone (Alf; 8 mg/kg IM) 2) ketamine alone (Ket; 20 mg/ kg IM) and 3) ketamine + dexmedetomidine (Ket+Dex; 4 mg/kg + 25 μg/kg IM) with reversal (atipamezole; 250 µg/kg IM). A total of 34 animals were evaluated, including 11 juveniles and 23 adults. Cardiopulmonary parameters such as indirect blood pressure, heart rate, respiratory rate, and SpO2 were measured, and blood was collected for blood gas analysis and a chemistry panel. To examine the efficacy of each sedation protocol, induction time, immobilization time, and recovery time were recorded. Subjective measures of quality and efficacy included quality of induction, pedal withdrawal reflex, palpebral reflex, muscle tension, rectal temperature, and quality of recovery. All 3 protocols successfully immobilized the animals and all animals recovered from sedation. Heart rates were highest among the Ket group and the lowest for the Ket+Dex group. On average, the Alf group was immobilized for twice as long as either the Ket or Ket+Dex groups. The Ket+Dex group had the fastest average recovery time and subjectively had the best quality of recovery. Based on these results, Ket+Dex is recommended over Alf or Ket alone for brief sedation of healthy galagos.

Abbreviations: BE, base excess; BP, blood pressure; CK, creatine kinase; DAP, diastolic arterial blood pressure; HCO3, bicarbonate;

HR, heart rate; MAP, mean arterial blood pressure; RR, respiratory rate; SAP, systolic arterial blood pressure; SpO2, oxygen- hemoglobin saturation; SO2, oxygen saturation; TCO2, total blood carbon dioxide

DOI: 10.30802/AALAS-JAALAS-19-000158

The Northern greater galago (Otolemur garnettii; also com- breathing.21,42,47,50 Recovery from ketamine sedation can be slow monly known as the small-eared galago, Garnett’s greater and animals frequently exhibit excessive salivation during the galago, or bush baby) is a nocturnal, arboreal, prosimian recovery period.25,47 In addition, the pH (3.5 to 5.5) of ketamine primate.6,51 Since the 1940’s Otolemur garnettii has been used is known to cause muscle damage in primates.4,15,32,37 When to study the evolutionary development of vision and soma- dexmedetomidine, an α-2 agonist, is used in combination with tosensation.20,38,52 ketamine, muscle relaxation is achieved and excessive saliva- Chemical restraint is often required to perform veterinary tion is usually limited.25,50,56 Because the combination requires and research procedures on laboratory primates. However, a smaller dose of ketamine to reach the same effect, and dexme- the literature devoted to the safety and/or efficacy of different detomidine is reversible with atipamezole, recovery is usually sedative and anesthetic drugs in prosimians is limited. Even faster.4,50,56 Dexmedetomidine administered alone causes a more scarce are publications pertaining specifically to galagos. decrease in heart rate, cardiac output, and blood pressure, in One study evaluated the cardiopulmonary and anesthetic ef- addition to respiratory depression.21,42,47,50 The combination of fects of sevoflurane anesthesia in Otolemur garnettii, finding it ketamine and dexmedetomidine usually results in increased a safe and effective drug for the induction and maintenance of heart rate and blood pressure, a net decrease in cardiac output, anesthesia.33 However, while inhalants can be useful in many and respiratory depression.21,36,42,47,51 situations, injectable drugs are often necessary to sedate animals In addition to ketamine and dexmedetomidine, alfaxalone within their primary enclosure. (alphaxalone) has also historically been recommended as an Ketamine is a commonly used sedative for many nonhuman injectable sedative in nonhuman primates.21,42,50 Alfaxalone primates, including prosimians.21,42,50,62 As a NMDA (N-methyl- is a neurosteroid which acts as a GABA (γ-aminobutyric acid) D-aspartate) antagonist, ketamine provides variable sedation agonist.21,31,42,47,50 The original veterinary formulation of with poor muscle relaxation.25,47 Ketamine is known to cause alfaxalone, branded Saffan, was a combination of alfaxalone an increase in heart rate, cardiac output, and blood pressure, and alphadolone, solubilized in Cremophor EL.31 Although in addition to a form of respiratory depression called apneustic historically used successfully to immobilize nonhuman primates, Saffan was removed from the market due to reports in dogs of severe adverse events that included histamine release Received: 12 Nov 2019. Revision requested: 09 Jan 2020. Accepted: 09 Mar 2020. 1 2 and anaphylactic reactions linked to the Cremophor EL vehi- Division of Comparative Medicine, and Department of Biostatistics,Vanderbilt Uni- 9,31,49 versity Medical Center, Nashville, Tennessee cle. A new veterinary formulation of alfaxalone, Alfaxan, *Corresponding author. Email: [email protected] was approved by the Food and Drug Administration in 2014.

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Formulated without alphadolone or Cremophor EL, Alfaxan of treatments = 3, and the block size = 12. Age was considered has since been used extensively and successfully in a wide the factor most likely to influence outcomes and therefore, 12 range of species including fish, amphibians, reptiles, birds, and juveniles (defined as animals age 1.5 to 3 y old) were assigned mammals, including nonhuman primates.4,7,31,63 Alfaxalone is within one of the 3 blocks and evenly distributed among the reported to have minimal effects on the cardiovascular system. treatment groups. During the study, 2 subjects were identified The most common effects include a transient reflex tachycardia with health concerns and one animal was noted to be pregnant. and a mild reduction in cardiac output reflected as a mild reduc- As these animals did not meet study criteria, their data were tion of blood pressure, with apnea sometimes reported after IV excluded, and alternate subjects were randomly selected to induction.31,40,41,47 replace them. Suitable alternate subjects were unavailable for 2 While the use of ketamine alone, ketamine and dexmedeto- original subjects and therefore the final population demographic midine combined, and alfaxalone alone have been investigated included 11 juveniles and 23 adults. Final treatment group sizes in other nonhuman primate species, including some prosim- were (1) ketamine alone (ket): n = 12, (2) alfaxalone (alf): n = 11, ians, no specific publications address their safety or efficacy and (3) ketamine and dexmedetomidine combined (ket+dex): n in Otolemur garnettii.24,63,64 The objective of this study was = 11. Age distribution by treatment group was (1) ket: 1.7 to 7.4 to evaluate and compare the efficacy and cardiopulmonary y, (2) alf: 1.5 to 6.3 y, and (3) ket+dex: 2.1 to 10.6 y. Sex ratio by effects of sedation with ketamine alone, ketamine combined treatment group was (1) ket: n = 7 males (M), 5 females (F), (2) with dexmedetomidine, and alfaxalone alone. We posed the alf: n = 2 M, 9 F, and (3) ket+dex: n = 8 M, 3 F. Weight distribu- following 3 hypotheses with regard to comparing the 3 drug tion by treatment group was (1) ket: 0.84 to 1.68 Kg, (2) alf: 0.84 regimens: 1) there would be no significant differences in ef- to 1.21 Kg, and (3) ket+dex: 0.88 to 1.53 Kg. ficacy, as measured by induction time, quality of induction, Sedation procedures. The study was performed over a pe- immobilization time, muscle tension, pedal withdraw reflex, or riod of 3 mo (December-February). All animals were sedated palpebral reflex; 2) there would be no significant differences in between 0839 and 1050 during their wake cycle. Food was the cardiopulmonary effects associated with sedation, as meas- withheld from each subject 16 to 18 h prior to sedation. The ured by heart rate (HR), respiratory rate (RR), blood pressure morning of the study, each animal was hand caught from the

(BP), rectal temperature, or blood parameters (pH, pCO2, BE home cage, placed in an empty pet carrier (Petmate, Arlington,

(base excess), HCO3 (bicarbonate), glucose, and lactate); and 3) TX), and weighed on a tared scale (Seca baby scale, Model alfaxalone would have a shorter recovery time, better recovery 354, Chino, CA). To administer the anesthetic injection, each quality, and less evidence of muscle damage as measured by animal was hand caught from the carrier and manually re- CK (creatine kinase), AST (aspartate aminotransferase), and strained while a second person administered the sedative into ALT (alanine aminotransferase). the cranial quadriceps muscle of the left leg using a 25-gauge needle. Total sedative dose volumes across all groups ranged Materials and Methods from 0.09 mL to 1.24 mL dependent on the animal’s weight. Animals. Animals were housed in an AAALAC-accredited Maximum intramuscular administration volumes per injection facility in accordance with the Guide for the Care and Use of Labo- site of 1.0 mL alfaxalone and 0.5 mL ketamine were approved ratory Animals,30 the Public Health Service Policy on Humane by the IACUC. The dose volume for the heaviest animal in the Care and Use of Laboratory Animals,44 and the Animal Welfare alfaxalone group exceeded this maximum allowable volume Act and Regulations.61 All research procedures involving ani- and therefore was divided into 2 equivalent doses (0.62 mL) mals were approved by the Institutional Animal Care and Use and administered in 2 sites in the cranial quadriceps muscle Committee at Vanderbilt University Medical Center (VUMC). of the left leg. This study enrolled 37 galagos scheduled for routine sedation Three sedation protocols were examined: (1) ketamine alone for tuberculosis testing and physical exams. All galagos were (100 mg/mL, KetaVed, Vedco, Saint Joseph, MO) at a dose of born in captivity at Vanderbilt University. The population was 20 mg/kg IM; (2) alfaxalone (10 mg/mL, Alfaxan Multidose, composed of 18 males and 19 females. Ages and body weights Jurox, North Kansas City, MO) at a dose of 8 mg/kg IM; or (3) ranged from 1.5 to 10.6 y old and 0.84 to 1.68 kg, respectively. a combination of ketamine and dexmedetomidine (0.5 mg/mL, Animals were housed in stainless steel NHP cages with a Dexdomitor, Zoetis, Parsippany, NJ) at a dose of 4 mg/kg + 25 squeeze-back mechanism (Lenderking, Millersville, MD) and μg/kg IM, respectively, combined in one syringe just prior to fitted with structural enrichment such as perches, resting sur- administration. After injection, the animal was returned to the faces, and/or hide tubes. Galagos were housed in rooms with a carrier where it was observed until immobilization occurred. 12:12-h reverse light cycle using red-light filters during the day. Once induced, the animal was removed from the carrier and Diet consisted of a commercial feline laboratory diet (LabDiet placed on a room temperature table without heat support. 5003, PMI Nutrition International, Brentwood, MO) supple- Galagos breathed room air without endotracheal intubation mented with fresh produce and foraging items (mealworms, throughout the procedure. All subjective and objective measures nuts, syrup, wheatgrass, etc.). Animals were also provided were recorded by a single, blinded observer who was not present manipulanda and auditory, visual, and olfactory enrichment in the room during the initial restraint or injection. on a rotational basis. Filtered municipal water was available A 12 mg/kg dose of alfaxalone has been successfully used to 4 ad libitum. Social housing was provided for compatible breed- sedate common marmosets (Callithrix jacchus). In a pilot study ing pairs and weanlings. Singly housed animals were always (data not shown) doses of 8 mg/kg, 10 mg/kg, and 12 mg/kg housed in rooms with visual, auditory, and olfactory contact were assessed in galagos. The 8 mg/kg dose provided consist- with other conspecifics. ent sedation and was chosen as the lowest effective dose for Randomization, masking, and data exclusion. Pregnant further exploration. The blinded observer did not participate and nursing animals, as well as animals with clinical health in the pilot study. concerns, were excluded from the subject pool. Subjects were Measures of induction and immobilization. The induction randomly assigned to 1 of 3 treatment groups using a permuted time was defined as the time between injection and the onset block design where the number of subjects = 36, the number of lateral recumbency. Immobilization time was defined as the

558 Evaluation of 3 sedation protocols in Otolemur garnettii

time between lateral recumbency and the first attempt of the collection were completed. Animals in the ketamine alone and animal to move their head. An observer who was blinded to alfaxalone groups received a 0.1 mL IM sham injection of 0.9% the treatment of the animal assessed the quality of induction by saline in the cranial quadriceps muscle of the right leg to main- using a scorecard (Figure 1). While immobilized, each animal tain a blinded study design and to control for the stimulation of was assessed by the observer for muscle tension, pedal with- reversal injection during immobilization. After administration drawal reflex, and palpebral reflexes using the scoring systems of the reversal agent or sham reversal, the animals were returned defined in Figure 1. Muscular tension was assessed every 3 min to the carrier, placed in lateral recumbency, and observed by the by lifting each limb of the animal while in dorsal recumbency observer until recovered. and allowing it to fall to the table. The pedal withdrawal reflex Recovery time was defined as the time from the animal’s first was assessed once, 8 to 14 min post induction, by applying attempt to move their head until they were able to maintain a hemostat (straight, smooth jaw, 5”) on the first clip for one sternal recumbency or a seated posture, unaided, for at least 120 second on the third digit, just above the nail of the left foot. seconds. The blinded observer assessed the quality of recovery The palpebral reflex was also assessed once, 8 to 14 min post by using a scorecard (Figure 1). Once recovered, animals were induction, by lightly touching the medial and lateral canthus transferred back into their home enclosure and monitored daily of the left and right eyes with a dry cotton swab. The variation for 3 d post sedation for any adverse consequences. in time for collecting these measures was due to the need to Statistical Analysis. Statistical analyses were performed us- complete the routine annual exam and the experimental pro- ing Prism 7.0 (GraphPad Software, San Diego, CA) and Stata/ cedures simultaneously. MP v16 (StataCorp LLC, College Station, TX). For continuous Measures of cardiopulmonary effects. Indirect systolic, mean, data, normality was assessed using the D’Agostino and Pearson and diastolic arterial blood pressure (SAP, MAP, and DAP) were normality test. Continuous parametric data were then analyzed measured oscillometrically (Surgivet, Advisor 3 Parameter Vital using a one-way ANOVA. One-way ANOVA analyses that Signs Monitor, Dublin, OH) using a cuff with a width that was resulted in significant differences between group means were approximately 40% of the circumference of the limb. The cuff further analyzed using posthoc Tukey multiple-comparison was initially placed on the lower right leg. If readings could tests. Nonparametric data was analyzed using the Kruskal–Wal- not be obtained, the cuff was moved to the right forearm or lis test. Significant differences between groups were identified tail base. Heart rate (HR) was measured using a Surgivet TPR using a Mann-Whitney test. Area under the curve (AUC) was Monitor (Model V3395, Dublin, OH), and confirmed via aus- calculated for repeated measures (HR, RR, SAP, DAP, and MAP), cultation. Oxygen-hemoglobin saturation (SpO2) was assessed averaged over unit time (AUC/min). These statistics were used by pulse oximetry (Surgivet TPR Monitor, Dublin, OH) with a as response features for longitudinal data and were analyzed transmission probe placed on the ear, hand, or foot. Respiratory as fixed-effects as for other continuous data.18 To analyze rate (RR) was determined by observing thoracic excursions and temperature loss over time, linear regressions were performed auscultation. Rectal body temperature was measured using a for each subject and the resultant individual slope coefficients digital thermometer (Model KD-1700, BestMed LLC, Golden, were then analyzed using a Kruskal–Wallis test, with significant CO). Cardiopulmonary measurements were recorded every 3 differences between groups identified using a Mann–Whitney min, beginning at the time of lateral recumbency and ending test. Pearson χ2 tests of association were run between the treat- when the animal exhibited purposeful movement of the head. ments and the subjective scoring for quality of induction, overall Blood was collected from the femoral vein using a 1 mL muscle tension, pedal withdrawal, and quality of recovery. We syringe and 25-gauge needle between 10 to 21 min post induc- used the Fisher protected least significant difference method to tion for measurement of blood gases and glucose, as well as assess multiple comparisons between treatments. Comparisons a complete blood count and chemistry panel. Blood for the between pairs of treatments were only assessed for significance blood gas analysis was placed in a Becton Dickinson lithium if the corresponding global test among all treatments was sig- heparin microtainer (BD, Franklin Lakes, NJ), and analysis nificant. Multivariate linear regression to adjust for age, sex, was performed using a portable clinical analyzer and CG4+ and weight was used to assess potential differences in these cartridges (iSTAT-1 clinical analyzer, Abbott Point of Care, variables by treatment group. Adjusted data were comparable

Princeton, NJ). CG4+ cartridges provided direct pH, PCO2, PO2, to unadjusted data; therefore age, sex, and weight did not af- and lactate measurements as well as calculated values for TCO2, fect the response to treatment. A P value of less than 0.05 was

HCO3, Base Excess (BE), and SO2. A veterinary glucometer (Al- regarded as statistically significant for all tests. phaTRAK 2, Zoetis, Parsippany, NJ), internally validated for use in galagos, was used at the time of blood collection to measure Results blood glucose. Blood for a complete blood count was placed Induction. Sedation was successfully induced in all animals. in a MiniCollect EDTA tube (Greiner Bio-One, Monroe, NC) A significant difference in induction time was detected between and kept refrigerated until processing (part of routine health treatment groups. (Figure 2; P = 0.0004) The induction time was monitoring, data not included). Blood for the chemistry panel significantly shorter for the ket group (mean = 2.1 min) com- was placed in a VACUETTE Z Serum Sep Clot Activator Tube pared with either the alf (mean = 4.9 min; P < 0.0001) or ket+dex (Greiner Bio-One, Monroe, NC) and kept at room temperature (mean = 3.5 min; P = 0.0130) groups. There was no significant dif- until processing. All blood was processed within 5 h of collection ference found between the alf and ket+dex groups (P = 0.0582). by a trained comparative pathology technician in the Vander- Quality of induction significantly varied between groups bilt Translational Pathology Shared Resource. Blood chemistry (Table 1, P = 0.013), with the quality of induction being sig- analysis was performed using a Vet Axcel Chemistry Analyzer nificantly less favorable for the alf group (mode = 2) compared (Alfa Wasserman Diagnostic Technologies, West Caldwell, NJ). with either the ket (mode = 1; P = 0.004) or ket+dex (mode = 1; Recovery. Animals receiving dexmedetomidine were dosed P = 0.016) groups. No significant difference was found between with 250 µg/kg of atipamezole IM (5 mg/mL, Antisedan, Zoetis, the ket and ket+dex groups (P = 0.227). The least favorable in- Parsippany, NJ) as a reversal agent in the cranial quadriceps duction score (score of 3) was observed only within the alf and muscle of the right leg after veterinary procedures and data ket+dex groups and was assigned most often due to significant

559 Vol 59, No 5 Journal of the American Association for Laboratory Animal Science September 2020

Figure 1. Quality of induction

Figure 2. Summary of induction, immobilization, and recovery times (means ± 95% CI). Superscripted lowercase letters indicate significant (P < 0.05) differences: a Different from Ket, b Different from Alf,c Different from Ket + Dex involuntary/uncoordinated activity during the induction phase. Immobilization. Immobilization times differed significantly By comparison, no animals in the ket group received a score between groups (Figure 2; P = 0.0009) with the alf group higher than 2. experiencing a significantly longer immobilization time (mean

560 Evaluation of 3 sedation protocols in Otolemur garnettii

Table 1. Summary of subjective scores. No. of animals per score (% of group) Treatment Score of 1 Score of 2 Score of 3 Quality of Induction Ketamineb 6 (50.0) 6 (50.0) 0 (0.0) Alfaxalonea,c 0 (0.0) 6 (54.5) 5 (45.5) Ket + Dexb 6 (54.5) 3 (27.3) 2 (18.2) Muscle Tension Ketamineb,c 1 (8.3) 4 (33.3) 7 (58.4) Alfaxalonea 8 (72.7) 1 (9.1) 2 (18.2) Ket + Dexa 9 (81.8) 2 (18.2) 0 (0.0) Pedal Withdrawal Reflex Ketamineb 11 (91.7) 1 (8.3) – Alfaxalonea,c 4 (36.4) 7 (63.6) – Ket + Dexb 9 (81.8) 2 (18.2) – Quality of Recovery Ketamineb,c 1 (8.3) 5 (41.7) 6 (50.0) Alfaxalonea 6 (54.6) 5 (45.4) 0 (0.0) Ket + Dexa 7 (63.6) 4 (36.4) 0 (0.0) Quality of induction, muscle tension, pedal withdrawal reflex, and quality of recovery are reported as [no of animals within treatment group per score (% of group)]. Palpebral reflex was absent across all subjects. N per treatment group was as follows: Ketamine n = 12, Alfaxalone n = 11, Ket + Dex n = 11. Superscripted lowercase letters indicate significant (P < 0.05) differences: a Different from Ket b Different from Alf c Different from Ket + Dex

= 40.0 min) compared with either the ket group (mean = 21.3 nificantly higher heart rates (mean = 301± 32 bpm, P < 0.0001) min; P = 0.0003) or the ket+dex group (mean = 23.2 min; P = than the alf or ket+dex groups (mean = 222 ± 23 bpm and 148 0.0041). No significant difference in immobilization time was ± 15 bpm, respectively). There was also a significant difference detected between the ket or ket+dex groups (P = 0.1842). between the alf and ket+dex groups (P = 0.0002) Results from subjective scoring of muscle tension and reflexes Respiratory rates ranged from 20 to 80 breaths per minute while animals were immobilized are shown in Table 1. There across all subjects and was significantly different between was a significant difference between groups for muscle tension groups (P = 0.0178). A significant difference (P = 0.0148) was (P = 0.002), with the overall muscle tension being significantly found between the ket (mean = 40 ± 10 breaths/min) and greater in the ket group (mode = 3) compared with either the ket+dex groups (57 ± 10 breaths/min). There were no significant ket+dex (mode = 1; P = 0.001) or the alf (mode = 1; P = 0.007) differences between alf (mean = 45 ± 8 breaths/min) and ket (P groups. There was no significant difference found between the = 0.6195) or alf and ket+dex (P = 0.1292) groups. ket+dex and alf groups (P = 0.302). The higher scores in the Blood pressure measurements, including SAP, DAP, and ketamine group were most often secondary to muscle rigidity MAP, were collected every 3 min and the average AUC per unit in more than one limb and/or opisthotonos. While a subset of time are depicted in Figure 3. The lowest blood pressures were animals in the alf group received scores of 3, the mode score for recorded in the alf group (mean MAP = 100 ± 9 mm Hg) and this group was 1. No animals in the ket+dex group received a the highest in the ket group (mean MAP = 126 ± 19 mm Hg). score higher than 2. Significant differences in SAP were identified between all groups Across all groups, the pedal withdrawal reflex was either (P = 0.0188). There was a significant difference in SAP between absent or delayed. There was a significant difference between the ket and alf groups (P = 0.0361) as well as between the alf and treatments for pedal withdrawal (P = 0.009). A delayed with- ket+dex groups (P = 0.0087). There was not a significant differ- drawal was observed most often in the alf group (7 of 11 ence in SAP between the ket and ket+dex groups (P = 0.4789). subjects), which was significantly different from the ket (1 of Significant differences in DAP were identified between groups 12 subjects; P = 0.005) and ket+dex (2 of 11 subjects; P = 0.030) (P = 0.0039). There was a significant difference in DAP between groups. No significant difference was found between the the ket and alf groups (P = 0.0105) as well as between the alf ket+dex and ket groups (P = 0.484). and ket+dex groups (P = 0.0013). There was not a significant The palpebral reflex was absent across all subjects in all difference in DAP between the ket and ket+dex groups (P = treatment groups. 0.9819). Significant differences in MAP were identified between Measures of cardiopulmonary effects. During immobilization, groups (P = 0.0036). There was a significant difference in MAP cardiopulmonary parameters, including HR, RR, and BP, were between both the ket and alf groups (P = 0.0077) and the alf and recorded every 3 min. Because of the variability in the length of ket+dex groups (P = 0.0015). There was no significant difference time animals were immobilized, the number of measurements in DAP between the ket and ket+dex groups (P = 0.7738). Blood between animals also varied. To account for this, the area under pressure readings could not be acquired in 5 subjects. All 5 of the curve for each animal was calculated and divided by the these animals were later determined to be in the ket group, had total immobilization time to acquire an average area under average heart rates greater that 260 bpm, and maximum muscle the curve per unit time. These values were then analyzed and tension scores of 3. compared. Values are reported as the mean ± 95% confidence Rectal temperatures were also recorded every 3 min. Due to interval, unless otherwise stated. variability in the length of time animals were immobilized, the Heart rates were significantly different between groups number of measurements between animals also varied. As such, (Figure 3; P < 0.0001) with animals in the ket group having sig-

561 Vol 59, No 5 Journal of the American Association for Laboratory Animal Science September 2020

Figure 3. Cardiopulmonary measurements. (A) Heart rate (B) Systolic arterial blood pressure (SAP) (C) Respiration rate (D) Mean arterial blood pressure (MAP) (E) Temperature loss over time (F) Diastolic arterial blood pressure (DAP), * = P < 0.05, ** = P < 0.01, *** = P < 0.001 linear regressions were calculated for each animal and the slope Blood gas analysis was performed in real time using an i- coefficient was used to assess the degree of temperature loss over STAT clinical analyzer. Measures of pH, PCO2, BE, HCO3, and time (Figure 3). Temperature measurements ranged from 94.5°F lactate can be found in Table 2. While blood pH measurements to 102.5°F, with a temperature loss over time ranging from 0.4 to for all animals ranged from 7.36 to 7.66, the means for each of 5.7°F. Significant differences were identified between groups (P the groups were the same (7.5; P = 0.1088). There were also

= 0.0012). Significant differences in temperature loss occurred minimal differences in blood pCO2 with a range of measure- between the ket and ket+dex groups (P = 0.0010) and between ments from 19.6 to 38.0 mm Hg. There was no significant the ket and alf groups (P = 0.0010). There were no significant difference in means between groups for pCO2 (P = 0.2312). Meas- differences between the alf and ket+dex groups (P = 0.8470). ures of HCO3 ranged from 15.4 to 30.6 mmol/L and significant differences were identified between groups (P = 0.0017). There

562 Evaluation of 3 sedation protocols in Otolemur garnettii

Table 2. Summary of blood values (mean ± 95% CI). All 3 sedation protocols proved efficacious in their ability Ketamine Alfaxalone Ket + Dex to immobilize galagos. Across groups, average induction time was less than 5 min, with the longest induction times occurring pH 7.5 ± 0.04 7.5 ± 0.02 7.5 ± 0.04 in the alf group and the shortest induction times occurring in pCO (mm Hg) 30.7 ± 3.5 27.7 ± 2.0 30.5 ± 2.8 2 the ket group. Induction quality varied significantly between HCO (mmol/L) b a,c b 3 24.9 ± 1.6 20.9 ± 2.7 25.6 ± 1.7 groups, with the quality of induction being significantly less BE (mmol/L) 2.2 ± 1.5b −2.7 ± 3.2a,c 2.3 ± 1.7b favorable in the alf group. These less favorable scores were due Lac (mg/dL) 1.5 ± 0.2b 4.3 ± 1.7a,c 1.6 ± 0.9b to significant involuntary and uncoordinated movement during Glu (mg/dL) 105 ± 6b,c 94 ± 26a,c 144 ± 26a,b induction that might represent a safety concern. This movement CK (U/L) 1259 ± 553c 782 ± 416 757 ± 723a was not simply “twitching”, as has been described in previous assessments of alfaxalone in multiple species.31 Rather this AST (U/L) 90.7 ± 27.8 62.5 ± 27.4 94.4 ± 71.6 movement included full body movements such as jumping and ALT (U/L) 78.3 ± 44.05 43.4 ± 18.4 55.2 ± 35.5 rolling. This increased activity may have represented the excite- Superscripted lowercase letters indicate significant (P< 0.05) differences: ment phase of anesthesia.26 This stage of anesthesia is usually a Different from Ket bypassed quickly with most modern sedatives and anesthetics. bDifferent from Alf A higher dose of alfaxalone than used in this study might reduce cDifferent from Ket + Dex the time required to bypass this stage. Alternatively, the large volume of alfaxalone required for intramuscular sedation may were significant differences between the ket and alf groups (P have been more slowly absorbed resulting in this excitatory = 0.0077) as well as the alf and ket+dex groups (P = 0.0025). phase. Further investigation could be accomplished in future Measurements of BE ranged from -10 to +7 mmol/L and sig- studies by increasing the dose and/or splitting the full dose nificant differences in BE were identified between groups (P = and administering it into more than one muscle. 0.0013). Significant differences were found between the ket and All 3 sedation protocols resulted in an adequate period of alf groups (P = 0.0033), as well as the alf and ket+dex groups immobilization to perform physical examination, blood col- (P = 0.0033). Lactate values ranged from 0.9 to 8.3 mg/dL with lection, and tuberculin skin testing. The immobilization time significant differences between groups (P = 0.0038). Significant achieved with alf was twice that of ket or ket+dex. The fastest differences were found between alf and ket (P = 0.0084) and alf recovery time occurred with ket+dex administration and was, and ket+dex (P = 0.0051). on average, one-third the time of recovery from ket or alf. Glucose was also measured in real time using a calibrated vet- However, atipamazole was administered to all animals in the erinary glucometer. Measurements ranged widely from 42 to 221 ket+dex group 15 to 25 min after induction; one of the benefits mg/dL and a significant difference was found between groups to using an α2 agonist is the ability to reverse the effects with (P = 0.0032). Significant differences were observed between ket atipamezole. This study did not aim to test the efficacy of theα 2 and ket+dex (P = 0.0091), ket and alf (P = 0.0487), and alf and reversal agent atipamezole in reducing recovery time, rather the ket+dex (P = 0.0043), with the highest glucose measurements aim of administering the atipamezole was to mimic what would being recorded in the ket+dex group and the lowest in the alf be used clinically. As such, the duration of immobilization and group (Table 2). recovery time of the ket+dex group without reversal was not Creatine kinase (CK), aspartate aminotransferase (AST), and assessed. Therefore, the duration of ket+dex immobilization alanine aminotransferase (ALT) were used as markers of pos- and recovery without atipamezole administration could have sible muscle damage (Table 2). No significant differences were resulted in recovery times similar to the ket and alf groups. found between groups for AST (P = 0.2371) or ALT (P = 0.2926). The quality of recovery was significantly different between all A significant difference between groups was found in CK (P = 3 treatments, with the ket+dex and alf groups displaying the 0.0497) between the ket and ket+dex groups (P < 0.0317). best quality of recovery and the ket group displaying the worst Recovery. Recovery times varied significantly between groups quality of recovery. Only animals in the ketamine group received (Figure 2; P = 0.0010), with the ket+dex group experiencing a scores of 3 (poor recovery). The poor quality of recovery scores significantly faster recovery time (mean = 10.4 min) than either assigned in the ket group were consistently due to significant the alf group (mean = 28.9 min; P = 0.0010) or ket group (mean uncoordinated activity during the recovery phase including = 30.9 min; P = 0.0005). The difference in means between the alf head pressing and paddling with the upper and lower extremi- and ket groups (means = 28.9 and 30.9 min, respectively) were ties such that safety was of concern. not significantly different (P = 0.8205). The quality of immobilization was assessed using muscle Quality of recovery differed significantly between groups (P tension, the pedal withdrawal reflex, and the palpebral reflex. = 0.003), being significantly better in the ket+dex (mode = 1) Muscle tension was significantly different between groups, and alf groups (mode = 1) than in the ket group (mode = 3; P = with the overall muscle tension being significantly greater in 0.005, P = 0.008, respectively). The quality of recovery was not the ket group than in either the ket+dex or the alf groups. This significantly different between the ket+dex and alf groups (P finding is consistent with reports of muscle rigidity in various = 0.665). The poor quality of recovery scores in the ket group other species sedated with ketamine.25,28,47 A delayed pedal were often due to marked uncoordinated activity during the withdrawal reflex (seen most often in the alf group) occurred recovery phase including head pressing and paddling with the in all 3 treatment groups, indicating that none of the protocols upper and lower extremities. tested are appropriate for surgical procedures. The palpebral reflex was absent, the eyes remained open, and eye position was Discussion central across all subjects in all treatment groups. The objective of this study was to evaluate and compare the The cardiopulmonary effects of each sedation protocol were efficacy and cardiopulmonary effects of sedation in Otolemur evaluated by assessing respiratory rate, heart rate, oscillometric blood pressure, SpO , and blood gases. Although a statistically garnettii with ketamine alone, ketamine combined with dexme- 2 detomidine, or alfaxalone alone. significant difference in respiratory rate was found between the

563 Vol 59, No 5 Journal of the American Association for Laboratory Animal Science September 2020 ket and ket+dex groups, this difference is unlikely to be clini- values in other nonhuman primate species suggests that the cally relevant. Heart rate and blood pressure were consistently values collected here are likely acceptable deviations from nor- 10,35,39,43,64 and significantly higher in the ket group. This difference was mal. The mean HCO3, BE, and lactate measurements in greater than expected but consistent with observations in other the alf group were consistently and significantly different from species.47,57 Heart rate was the lowest in the ket+dex group, both the ket and ket+dex groups. These significant differences 21,47 which is also consistent with observations in other species. were due to the HCO3 measurements frequently being lower, Transient, physiologic heart murmurs were auscultated in 2 the BE being most often negative, and the lactate being higher of the original subjects in the ket+dex group (their data was in the alf group. These differences suggest a metabolic acidosis excluded from all analyses). Heart murmurs are reported as in the alf group. However, the consistency in blood pH supports possible adverse effects of dexmedetomidine administration the conclusion that healthy galagos can adequately compensate in cats and caution is advised in patients with cardiovascular for any minor, transient metabolic or respiratory acidosis or al- disease per the Dexdomitor package insert.65 kalosis that might occur with administration of these sedatives. Several studies have validated the use of oscillometric blood Glucose measurements ranged widely, with significant dif- pressure measurements in other species.12,17,54,60 However, oscil- ferences observed between all 3 groups. The highest glucose lometric blood pressure measurements have well-documented measurements were recorded in the ket+dex group and the limitations, including movement and increased muscle rigid- lowest in the alf group. Conflicting data in the literature sug- ity.23,27,59 In the present study, blood pressure readings could not gests transient hyper- or hypoglycemia may occur with the be obtained in 5 subjects in the ket group, perhaps due to the administration of ketamine or α2 agonists.2,8,11,16,46,53 Limited muscle tension and frequent movement of these animals (all 5 data are available about the effects of alfaxalone on serum of these subjects had muscle tension scores of 3). In addition, glucose. In addition, transient hyperglycemia may result from while the majority of measurements were taken with the blood the stress associated with hand-capture and injection.14,22,45 As pressure cuff placed on the lower right leg, several subjects had such, caution should be used when administering sedatives to blood pressure readings taken from the right forearm or tail base any prediabetic or diabetic animals. due to the inability to obtain a reading with the cuff placed on CK, AST and ALT are commonly used to assess muscle dam- the lower right leg. The lack of consistency in placement may age in multiple species.34 A statistically significant difference also represent a shortcoming of the present study.1,23,59 While was found between the ket and ket+dex groups, with the ket- intraarterial blood pressure monitoring is considered the ‘gold dex group having a lower average CK. Ketamine is associated standard’ for accurate blood pressure measurement, the inva- with volume-dependent tissue damage in many species, likely siveness of and time required to place arterial lines made this due to its acidity.5,15,58 Alfaxalone has a neutral pH (6.5 to 7) method impractical for this study and for routine clinical use. but requires a markedly higher volume of drug to be effective Another limitation of our study is that we did not measure than either ket or ket+dex. While no significant differences in physiologic parameters in conscious animals, and there are no AST or ALT were found between groups, a better assessment published blood pressure reference ranges for Otolemur garnettii. would have included repeat blood sampling within 24 to 48 h Therefore, we have no baseline values for comparison with those after sedation, as release of AST and ALT in response to muscle obtained under sedation. To obtain this data, surgical placement damage may require longer than 6 h3,48 While we cannot exclude of monitoring equipment would be required. At our institu- an effect of hand-capture on the measures of muscle damage, tion, a blood pressure of 120/80 mm Hg is commonly used as we can conclude that the larger volume of alfaxalone required a reference in several nonhuman primate species, including for intramuscular sedation is unlikely to be any more damag- galagos. When using noninvasive blood pressure monitoring, ing to muscle than intramuscular administration of ketamine. the trend observed over time within an individual is most often In conclusion, ketamine combined with dexmedetomidine more clinically relevant than the actual numerical value. In is recommended for the sedation of healthy galagos without the present study, the differences in blood pressure that were evidence of heart disease for brief, minor procedures. observed between treatment groups are consistent with the published literature on the effects of each drug on blood pres- 21, 31, 40, 41, 42, 47, 50 Acknowledgments sure. We thank the Division of Comparative Medicine and the Vanderbilt

SpO2 data was collected (data not shown) but regarded as University Medical Center Laboratory Animal Medicine Residency inaccurate due to consistently low readings (55% to 89%). These Program for funding this work. We extend sincere gratitude to Amy readings did not coincide with visual assessment of the animals MacKenzie for her technical assistance. We thank Mary Feurtado for her or their respiratory rates. Excessive motion, skin pigmentation, galago expertise and Dr Jon H. Kaas for access to the animals (funded in ambient light, and sensor placement are important factors that part by NIH grant EY 002686). We acknowledge the Translational Pathol- ogy Shared Resource supported by NCI/NIH Cancer Center Support may influence SpO readings.13,19,29 In this study, motion artifact 2 Grant 5P30 CA68485-19, the Vanderbilt Mouse Metabolic Phenotyping and skin pigmentation of the hands, feet, and ears where the Center Grant 2 U24 DK059637-16 and Shared Instrumentation Grant pulse oximeter was placed, likely contributed to inaccurate SpO2 S10 OD023475-01A1. measurements. The possibility that these drugs could cause hypoxemia cannot be excluded and further investigation could be accomplished by repeating the study with the addition of References 1. Acierno MJ, Domingues ME, Ramos SJ, Shelby AM, da Cunha supplemental oxygen. AF. 2015. Comparison of directly measured arterial blood pressure Venous blood gases including pH, pCO2, HCO3, BE, and at various anatomic locations in anesthetized dogs. Am J Vet Res lactate were assessed. Blood gases are species specific and dif- 76:266–271. https://doi.org/10.2460/ajvr.76.3.266. ferent sedatives or anesthetics, along with the type of procedure 2. Ambrisko TD, Hikasa Y. 2002. Neurohormonal and metabolic being performed, can alter blood gas results.55 A limitation of effects of medetomidine compared with xylazine in beagle dogs. this study is that published blood gas reference ranges are not Can J Vet Res 66:42–49. available for Otolemur garnettii and as such, limited conclusions 3. Arun P, Oguntayo S, Alamneh Y, Honnold C, Wang Y, Vali- yaveettil M, Long JB, Nambiar MP. 2012. Rapid release of tissue can be drawn from our data. Review of the published blood gas enzymes into blood after blast exposure: potential use as biological

564 Evaluation of 3 sedation protocols in Otolemur garnettii

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&lt;I&gt;Otolemur Garnettii&lt;/I

<I>Otolemur Garnettii</I