Short term pharmacological immobilization in macaque monkeys

Veterinary Anaesthesia and Analgesia, 2011, 38, 490–493

doi:10.1111/j.1467-2995.2011.00637.x

SHORT COMMUNICATION

Short term pharmacological immobilization in macaque monkeys Martin Votava*, Ladislav Hess , Jitka Schreiberova´à, Jirˇ´ı Ma´lek§ & Karel Sˇtein– *Department of Pharmacology, 2nd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic  Institute for Clinical and Experimental Medicine, Laboratory of Experimental Anaesthesiology, Prague, Czech Republic àDepartment of Anaesthesiology and Intensive Care Medicine, Faculty of Medicine and University Hospital Hradec Kra´love´, Charles University in Prague, Hradec Kra´love´, Czech Republic §Department of Anaesthesiology and Critical Care Medicine, 3rd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic –Military Health Insurance Company, Prague, Czech Republic

Correspondence: Martin Votava, Department of Pharmacology, 2nd Faculty of Medicine, Charles University in Prague, V U´valu 84, Prague 5, 15 006 Czech Republic. E-mail: [email protected]

Abstract Objective To develop a safe and effective immobilization protocol in rhesus monkeys, which is not based on dissociative anaesthetic agent. Study design Prospective, randomised, experimental trial. Animals Twenty rhesus monkeys, weighing 2.6– 8.0 kg, 1–3 years of age, of both sexes. Methods The monkeys received 50 lg kg)1 medetomidine, 0.25 mg kg)1 midazolam and 5 lg kg)1 fentanyl with 150 IU hyaluronidase intramuscularly (IM). The animals were closely observed for behavioural changes and reaction to sound stimulus. Pulse rate and oxygen saturation of haemoglobin (SpO2) were monitored every 5 minutes, for 20 minutes. After this period, 250 lg kg)1 atipamezole or a placebo was administered IM and behavioural changes were closely observed. Results Full immobilization was observed after mean 269 ± SD 116 seconds. Ten minutes after injection mean arterial oxygen saturation of haemoglobin was 94 ± 4%, but did not fall significantly further. The median pulse rate was 116 beats minute)1 5 minutes after the administration of the drug. This level 490

further decreased to a median level of 108 beats minute)1 20 minutes after the drug’s administration. The median time to recover from immobilization was significantly shorter after atipamezole administration when compared to placebo (2.7 versus 55 minutes). All animals awoke smoothly and no side effects such as vomiting or agitation were observed. Conclusions Short term and reversible pharmacological immobilization was achieved using combination of midazolam, medetomidine, and fentanyl. Clinical relevance The present study demonstrates that 20-minute pharmacological immobilization with a combination of midazolam, medetomidine, and fentanyl is feasible in rhesus monkeys with minimal effect on heart rate. Keywords: alpha2-adrenergic agonist, anaesthesia, fentanyl, medetomidine, midazolam, primate.

Introduction Reliable sedation without arousal to external stimuli is essential for the safe handling and treatment of primates. Alpha2-adrenoceptor agonists, such as medetomidine, play an important role in the pharmacological immobilization of primates. The

Short term reversible immobilization in macaques M Votava et al.

activation of alpha2-adrenoceptors mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia (Sinclair 2003). In primates the most commonly described immobilization protocols are based on the administration of dissociative anaesthetic agents including ketamine, often combined with medetomidine (Sun et al. 2003) and the combination of tiletamine with zolazepam (Bentson et al. 2003). In our laboratory we have described the combined immobilization effect of medetomidine or its derivative naphthylmedetomidine with midazolam and ketamine in anthropoid apes (Hess et al. 2010) and rhesus monkeys (Votava et al. 2009). However, the dissociative anaesthetic agents are known to cause many adverse reactions, among which salivation and hypertonic reactions are the most disturbing. The aim of this study was to develop a safe and effective short-term immobilization protocol in rhesus monkeys that is not based upon a dissociative agent and can be reversed. Based on published results (Henke et al. 2004) and our experiments we decided to use the combination of midazolam, medetomidine and fentanyl, with atipamezole as a reversal agent. Materials and methods Subjects Twenty rhesus monkeys (Macaca mulatta; Biotest Kona´rovice, Czech Republic), weighing 2.6–8.0 kg, aged 1–3 years and of both genders (8 females, 12 males) were used. The monkeys were housed in individual cages (3 · 2 · 3 m) with an outdoor enclosure. Room temperatures were 20–22 C, relative humidity 40–65% and cages were under room lighting, with lights on from 0600 to 1800 hours. Food and water were available ad libitum. Drugs Medetomidine (Domitor; Pfizer, Czech Republic) 50 lg kg)1; midazolam (Dormicum; Roche, Czech Republic) 0.25 mg kg)1; fentanyl (Fentanyl-Janssen; Janssen-Cilag, Czech Republic) 5 lg kg)1; hyaluronidase (Hylase Dessau; Riemser Azrneimittel, Germany) 150 IU were administered via one intramuscular (IM) injection into the musculus deltoideus and atipamezole (Antisedan; Orion Corporation, Finland) 250 lg kg)1 or a saline

placebo was administered via another IM injection into the musculus deltoideus. Hyaluronidase was added to the immobilising combination to enhance the absorption and distribution of the injected drugs. Procedures Experiments were approved by the Expert Committee for the Protection of Experimental Animals of IKEM and were performed in accordance with the Animal Protection Act of the Czech Republic (No. 246/1992 Sb). The animal was placed in the observational cage and was temporarily immobilized using the retractable rear wall of the cage whilst the injections were administered. This was necessary due to the naturally aggressive behaviour of rhesus monkeys. After administering the drugs, the animals were observed closely for behavioural changes, such as sedation, ataxia, tactile reaction, loss of aggressiveness (reaction of the animal to approaching hand and touch) and reaction to sound stimulus (clap of the hands). As soon as we were able to remove the monkey from the cage (after the loss of aggressiveness), we began measuring pulse rate (PR) and the oxygen saturation of haemoglobin (SpO2) on a forelimb digit using a pulse oximeter (Nonin 80 500 V) which was placed on a forelimb digit for a period of 20 minutes. After this period atipamezole (n = 10) or placebo (n = 10) were administered and the recovery times were measured. The immobilization onset time was defined as the time between the injection and the moment of the animal sinking to the cage floor in a supine or lateral position. The loss of grip reflex was tested once this had occurred. The time of recovery was defined as the ability of the monkey to sit in the cage. Side effects such as respiratory depression, bradycardia, excitation activities or vomiting were recorded throughout the whole study period (immobilization and recovery phases) if and when they occurred. Data analysis Following testing for normality of data distribution, the SpO2 was evaluated using the one-way repeated measures ANOVA. The PR was analyzed using Friedman Repeated Measures Analysis of Variance on Ranks. The time of recovery from the immobilization was evaluated using the Mann–Whitney

 2011 The Authors. Veterinary Anaesthesia and Analgesia  2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38, 490–493

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Short term reversible immobilization in macaques M Votava et al.

Rank Sum Test. All statistical tests used two-tailed criteria, with a significance level of p < 0.05. Results are given as mean ± standard deviation, or median (range) as appropriate. Results The mean onset of immobilization with a loss of aggressiveness was observed after 74 ± 16 seconds. Full immobilization was observed after 269 ± 116 seconds. At the moment of immobilization there were no visual responses (awaking, gestures or movement) nor response to hand clapping and tactile stimuli. Grip reflexes were lost after 288 ± 127 seconds. The mean SpO2 at the first measurement point (Table 1) was 95 ± 3%, and this level was maintained during the whole immobilization period (p = 0.61). In two animals only did the oxygen saturation drop below 90%. The median (range) PR, (Table 1) was 116 (94–140) beats minute)1 at the first measurement point, then continued to fall significantly over time (p < 0.001). The median time to recover from the immobilization was significantly shorter after atipamezole administration than after the placebo (2.7 [1.5–5.8] versus 55 [20–118] minutes; T = 155, p £ 0.001). All animals awoke smoothly and no side effects such as vomiting or agitation were observed. Discussion The present study demonstrates that pharmacological immobilization with a combination of midazolam, medetomidine, and fentanyl is feasible in primates for the 20 minute post drug period evaluated. This combination is able to easily induce relatively long lasting immobilization with the possibility of a relatively rapid reversal using atipamezole. The onset of immobilization shorter than 5 minutes is fast enough to be routinely used in veterinary practice.

A combination of 30 lg kg)1 medetomidine and 0.3 mg kg)1 midazolam without opioids was used to induce chemical restraint in Japanese macaques (Miyabe et al. 2001). Although effective in most, immobilization was not achieved in all monkeys indicating that either drug dosages must be increased or additional drugs given in order to achieve complete immobilization in all animals. When medetomidine was used alone in doses as high as 120 lg kg)1, all monkeys became sedated but were easily aroused by external stimuli and only some animals assumed a recumbent position, indicating that the single use of medetomidine was insufficient (Miyabe et al. 2001). Pulley et al. (2004) investigated midazolam given as a single agent to macaques, and found that it provided only mild analgesia with no immobilization. Thus the additive and synergistic effects of different hypnotic drugs appear to be necessary in order to be effective. We decided to modify these previously published protocols, based on our preliminary results and other published studies, where the combination of benzodiazepines, opioids and an alpha2-adrenoceptor agonist was used. We used subanaesthetic doses of fentanyl and of midazolam. The recommended dose of fentanyl (in combination with other agents) to induce anaesthesia in primates is approx. 10– 25 lg kg)1 (Popilskis & Kohn 1997), while for midazolam the dose ranges between 0.5 to 1 mg kg)1 (Pulley et al. 2004). The dosage of 50 lg kg)1 medetomidine was chosen as the mean effective dose for sedation in primates. In the present study, the addition of 5 lg kg)1 fentanyl plus doses of medetomidine slightly higher than those previously published appeared to be superior and ensured immobilization in all monkeys. Although fentanyl was not antagonized, recovery times were faster than reported by Miyabe et al. (2001). This rapid recovery may be explained by the fact that we used a higher dose of atipamezole.

Table 1 Pulse rate and arterial haemoglobin oxygen saturation in rhesus monkeys (n = 20) during the first 20 minutes after IM drug administration. The drug combination given was medetomidine 50 lg kg)1, midazolam 0.25 mg kg)1, fentanyl 5 lg kg)1 and hyaluronidase 150 IU. Results are given as mean ± SD. For SpO2 (arterial oxygen saturation of haemoglobin) and median (range) for pulse rate

Time after drug administration, minutes SpO2 (%) Pulse rate (beats minute)1)

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5 95 ± 3 116 (94–140)

10 94 ± 4 112 (91–138)

15 94 ± 3 107 (86–129)

20 94 ± 4 108 (88–127)

 2011 The Authors. Veterinary Anaesthesia and Analgesia  2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38, 490–493

Short term reversible immobilization in macaques M Votava et al.

In a published report where anaesthetic doses of midazolam and fentanyl were used, a significant drop in the respiratory rate, with a short period of apnea in some animals was observed (Henke et al. 2004). However, the dose used in their study was chosen to provide anaesthesia, whilst our protocol was designed to induce immobilization. In our study, the doses of midazolam and fentanyl used were four-times lower than those used by Henke et al. (2004), and explain why the cardiorespiratory functions were only marginally affected. The effect on PR was minimal assuming that baseline pulse rates were around physiological levels of 150 beats minute)1 (Olberg 2007). The SpO2 was also affected, but to a smaller extent compared to most immobilizing combinations e.g. medetomidine, ketamine (Henke et al. 2004). As a limitation of this study, no other cardiovascular parameters such as blood pressure monitoring were taken, in order to disturb the animals as little as possible. If residual analgesia is to be maintained, then only one of the two agents, medetomidine or fentanyl should be reversed (Henke et al. 2004). Our previous results (Hess et al. 2010) and published studies in other species have shown that medetomidine alone induces a long period of sedation with a decrease in the respiratory rate and of arterial oxygen saturation (Sinclair 2003). Therefore generally it is recommended to reverse the medetomidine (Sun et al. 2003; Henke et al. 2004). Fentanyl is a short acting agent and midazolam in the low dose used in our study does not induce sedation, and therefore we decided to reverse only the effect of medetomidine. This study shows that recovery time after administration of atipamezole was 2.7 (1.5–5.8) minutes; awaking was rapid and smooth, and no specific antagonists of benzodiazepines or opioids were required. In conclusion, this study demonstrates a novel, effective, short-term and reversible immobilization method for rhesus monkeys which does not involve the use of dissociative anaesthetic agents and is based on the combination, of an alpha2-adrenoceptor agonist, a benzodiazepine and an opioid. Our data collection consisted only of PR and SpO2, but within the measurements obtained, physiological effects associated with the technique were clinically acceptable, although administration of oxygen might be advisable to reduce the fall in haemoglobin oxygen saturation.

Acknowledgement This work was supported by the grant IGA NT 11284–4/2010 from the Czech Ministry of Health. References Bentson KL, Capitanio JP, Mendoza SP (2003) Cortisol responses to immobilization with Telazol or ketamine in baboons (Papio cynocephalus/anubis) and rhesus macaques (Macaca mulatta). J Med Primatol 32, 148– 160. Henke J, Baumgartner C, Roltgen I et al. (2004) Anaesthesia with midazolam/medetomidine/fentanyl in chinchillas (Chinchilla lanigera) compared to anaesthesia with xylazine/ketamine and medetomidine/ketamine. J Vet Med A Physiol Pathol Clin Med 51, 259–264. Hess L, Votava M, Schreiberova J et al. (2010) Experience with a naphthylmedetomidine-ketamine-hyaluronidase combination in inducing immobilization in anthropoid apes. J Med Primatol 39, 151–159. Miyabe T, Nishimura R, Mochizuki M et al. (2001) Chemical restraint by medetomidine and medetomidinemidazolam and its reversal by atipamezole in Japanese macaques (Macaca fuscata). Vet Anaesth Analg 28, 168– 174. Olberg RA (2007) Monkeys and gibbons. In: Zoo Animal & Wildlife: Immobilization and Anesthesia. West G, Heard D, Caulkett N (eds). Blackwell Publishing, Oxford, UK. pp. 375–386. Popilskis SJ, Kohn DF (1997) Anesthesia and analgesia in nonhuman primates. In: Anesthesia and Analgesia in Laboratory Animals. Kohn DF (ed). Academic Press, New York, NY, USA. pp. 234–254. Pulley AC, Roberts JA, Lerche NW (2004) Four preanesthetic oral sedation protocols for rhesus macaques (Macaca mulatta). J Zoo Wildl Med 35, 497–502. Sinclair MD (2003) A review of the physiological effects of alpha2-agonists related to the clinical use of medetomidine in small animal practice. Can Vet J 44, 885– 897. Sun FJ, Wright DE, Pinson DM (2003) Comparison of ketamine versus combination of ketamine and medetomidine in injectable anesthetic protocols: chemical immobilization in macaques and tissue reaction in rats. Contemp Top Lab Anim Sci 42, 32–37. Votava M, Hess L, Schreiberova J et al. (2009) The effect of the novel partial alpha2-adrenoceptor agonist naphthylmedetomidine on the basic cardiorespiratory parameters and behavior in rhesus monkeys. J Med Primatol 38, 241–246. Received 6 December 2010; accepted 5 January 2011.

 2011 The Authors. Veterinary Anaesthesia and Analgesia  2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 38, 490–493

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