Transnasal administration of a combination of dexmedetomidine, midazolam and butorphanol produces deep sedation in New Zealand White rabbits

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1 Veterinary Anaesthesia and Analgesia, 2016, 43, doi: /vaa SHORT COMMUNICATION Transnasal administration of a combination of dexmedetomidine, midazolam and butorphanol produces deep sedation in New Zealand White rabbits Bruna Santangelo*, Fabiana Micieli*, Tiziana Mozzillo, Fabiana Reynaud, Francesco Marino, Luigi Auletta & Giancarlo Vesce* *Department of Veterinary Medicine and Animal Productions, University of Napoli Federico II, Napoli, Italy Clinica veterinaria del Chianti, Greve in Chianti, Italy Clinica veterinaria Sannio, Sant Angelo a Cupolo, Benevento, Italy Clinica veterinaria Animalia srls, Aversa, Caserta, Italy IRCCS SDN, Napoli, Italy Correspondence: Bruna Santangelo, Department of Veterinary Medicine and Animal Productions, Via Delpino 1, Napoli, Italy. bruna.santangelo@unina.it Abstract Objective To study the sedative and cardiorespiratory effects of transnasal (TN) administration of a combination of dexmedetomidine (DEX), midazolam (MID) and butorphanol (BUT) administered through a nasal catheter to rabbits undergoing diagnostic procedures. Study design Descriptive cross-sectional experimental study. Animals Eight healthy New Zealand White rabbit does (12 1 months old, kg). Methods DEX (0.1 mg kg 1 ), MID (2 mg kg 1 ) and BUT (0.4 mg kg 1 ) were mixed (DMB) in a syringe and applied to the rabbits nasopharyngeal mucosa after the accurate catheterization of one nostril. The onset, duration and quality of effects including analgesia were scored using a numeric rating scale of sedation for rabbits. Continuous monitoring of vital parameters was performed via clinical and multiparametric recording. Physiological variables were explored using repeated measures ANOVA for parametric data or Friedman s test for non-parametric data. Tukey s or Dunn s post hoc multiple comparisons test was used depending on normality. The statistical significance was set at p < Results Loss of the righting reflex, deep sedation and profound analgesia ensued simultaneously at minutes after DMB administration. These effects lasted 45 minutes before subsiding into moderate sedation, which lasted for an additional 25 minutes. Residual central nervous system impairment persisted up to 100 minutes. Blood pressure dropped progressively over time by 50%, whereas respiratory frequency decreased by 70%, consistent with moderate hypoxemia and hypercarbia. Conclusion and clinical relevance The TN route is a reliable and effective means for administration of DEX, MID and BUT to rabbits. The overall profound sedative effects and analgesic proprieties of the DMB combination can be selectively reversed depending on the needs of the procedure. Oxygen supplementation and careful monitoring are mandatory even in healthy subjects. The DMB protocol should be cautiously used in rabbits with cardiovascular or respiratory deficiencies. Keywords butorphanol, dexmedetomidine, midazolam, nasal, rabbit. 209

2 Introduction In rabbits, anaesthetic induction using single parenteral drugs requires high doses, leading to apnoea, hypoxemia, inability to control anaesthetic depth and at times prolonged recovery. Ketamine alone produces inconsistent anaesthetic and analgesic effects in rabbits while its combination with midazolam produces a light to medium plane of anaesthesia. The addition of butorphanol to the ketamine/ midazolam combination increases the duration of anaesthesia (Hedenqvist et al. 2002). More recently, a combination of midazolam fentanyl medetomidine, which can be reversed by specific antagonists, has been reported as an effective anaesthetic in rabbits, producing surgical anaesthesia (Henke et al. 2005). The dextrorotatory enantiomer of medetomidine, dexmedetomidine (DEX), is a highly selective alpha- 2 adrenergic receptor agonist. The respiratory depressant effects of DEX have been studied in rabbits by Nishida et al. (2002), who reported a reduction in respiratory frequency ( f R ) and tidal volume (V T ) unaccompanied by profound hypoxemia or hypercapnia. The water-soluble formulation and powerful sedative effect of DEX are suitable requisites for mucosal absorption; in fact, DEX can be effectively administered via the intranasal route in humans and animals (Schnellbacher et al. 2012). Midazolam (MID) produces intense tranquilization in rabbits, along with good muscle relaxation and synergistic effects with other anaesthetics or opioids. The pharmacological features of MID favour its oral and intranasal absorption, as shown in children and dogs (Eagleson et al. 2012). In rabbits, intranasal administration of 2 mg kg 1 MID has an onset time of 3 minutes and duration of approximately 25 minutes (Robertson & Eberhart 1994). Although MID exerts little depressant effects on the cardiovascular and respiratory systems, in rabbits, the combination of MID with other anaesthetic agents requires careful monitoring of respiratory function (Henke et al. 2005; Hedenqvist et al. 2013). Butorphanol tartrate (BUT) is a powerful agonistantagonist opioid analgesic with minimal cardiorespiratory side effects in rabbits (Portnoy & Hustead 1992), although its combination with MID causes mild hypoxemia (Schroeder & Smith 2011). The water-soluble formulation of butorphanol and its high concentration are good candidates for transmucosal absorption, as shown by its intranasal administration in humans. Given the pharmacological qualities of DMB and the remarkably deep sedation observed in captive hares after its transnasal (TN) administration (personal observations), we were inspired to observe its sedative and cardiorespiratory effects in rabbits. The choice of the TN route rather than the intranasal one was as a result of the large volume of solution that would need to be administered. We hypothesized that the TN administration of the DMB combination would induce profound sedation with tolerable cardiorespiratory depression in New Zealand White rabbits. Materials and methods Animals and housing This study was approved by the welfare regulation committee (CESA) of the University Federico II of Naples under project license number 2011/ All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No , revised 1996). The study was undertaken, between and hours, in a mobile anaesthesia lab set up on the farm where the animals were born and raised. Eight, American Society of Anesthesiologists physical status (ASA) I, New Zealand White rabbits (brood females) with a mean SD of 12 1 months and body weight of kg were randomly selected from a rabbit colony free from common rabbit pathogens. Twenty-four hours prior to the execution of the study, the animals were assessed healthy based on physical, haematological and biochemical examinations, identified using ear tags and transferred to individual flat-deck cages ( cm) for acclimatization in their own barn (temperature, C; humidity, 40 60%; 12:12 hour light dark cycle). A commercial pellet diet (Liverini pellets, Italy), autoclaved hay and water were available ad libitum. Study design At the time of the study, one assistant gently restrained each rabbit in a sitting position at the edge of the examination table by means of a fabric towel with the head exposed. The rectal temperature (T C), heart rate (HR), respiratory frequency ( f R ) and pulse Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

3 oximetry (SpO 2 ) were quickly measured. A preloaded syringe containing 0.1 mg kg 1 (0.2 ml kg 1 ) DEX (Dexdomitor 0.05%; Pfizer Italia srl, Italy), 2mgkg 1 (0.4 ml kg 1 ) MID (Midazolam 0.5%, IBI S.p.A., Italy) and 0.4 mg kg 1 (0.04 ml kg 1 ) BUT (Dolorex 1%, Intervet Production srl, Italy), reaching a total volume of 0.64 ml kg 1, was quickly hooked to a premeasured lubricated (Luan, Molteni & C. srl, Italy) catheter [Levin probe 2.00 mm (CH 6)], which was smoothly inserted through one nostril up to the medial angle of the ipsilateral eye, achieving drug deposition at the level of the nasopharyngeal mucosa (T-0). After DMB administration, each subject was quickly released to observe the effects of the drugs. The level of sedation was assessed by recording the rabbit s posture, the loss of the righting reflex (T-1), the palpebral reflex and reactions to other stimuli using a modified numeric rating scale (0 12) for rabbits (Appendix S1) (Raekallio et al. 2002). This individual sedation score (SS) was assessed every 5 minutes by the same operator in all rabbits and was classified as insufficient (0 3), moderate (4 7) or deep (8 12). Analgesia was scored by the pedal withdrawal reflex (PWR) on a 0 2 scale as part of the sedation score. An ultrasound examination was performed on anaesthetized rabbits. From T-1 (time = 0 minutes), the subjects were placed in lateral recumbency on a heating pad (Gaymar pump-tp500). After ear vein and ear artery catheterization, 10 ml kg 1 0.9% saline were infused intravenously (IV) throughout the study. The following parameters were continuously recorded: electrocardiogram (ECG); HR; f R ; SpO 2 ; end-tidal carbon dioxide (PE CO 2 ); T C; and indirect systolic, diastolic and mean arterial pressure (SAP, DAP and MAP) from T-1 to T-45 using a multiparametric monitor (Mindray Express PM-9000, Mindray Medical S.r.l., Italy). SpO 2 was measured by placing the oximeter probe at the base of one ear; PE CO 2 was recorded using Microstream technology by placing the tip of the sampling line directly into the vestibule of one nostril, while SAP, DAP and MAP were monitored by placing the blood pressure cuff above the carpus. Whenever O 2 saturation fell below 90%, a 2 L minute 1 oxygen flow was brought next to rabbit s nares. Arterial blood-gas analysis was performed at T-20 in all subjects, then at different intervals in different subjects. No attempt to intubate the spontaneously breathing animals was made. At T-45, all subjects were moved back to their holding cages, where the sedation level was assessed every 10 minutes until complete recovery. Statistical analysis All numerical data were recorded using a computerized spreadsheet (Microsoft Excel 2011) and imported into a program for statistical analysis (JMP 8.0.2, 2009; SAS Institute Inc., USA). Trends in the physiological variables over time were explored using a repeated measures analysis of variance (ANOVA) with Greenhouse Geisser correction for parametric data or Friedman s test for non-parametric data. Tukey s or Dunn s post hoc multiple comparisons test was used depending on normality. The normality of the data was determined with a Shapiro Wilk W test. The statistical significance was set at p < Results Normally distributed data are expressed as the mean SD, whereas non-parametric data are reported as the median (range), as summarized in Table 1. The mean latency to loss of righting reflex (T-0 T- 1) was minutes. T-1 was reached after 4.2 minutes in one rabbit. The sedation score was classified as deep from T-1 (11; 8 12) to T-45 (8; 5 12) and moderate from T- 45 to T-70 (4; 1 6). From T-70 onward, the sedation level was insufficient (median < 4), leading to complete recovery at variable intervals. The rabbits were all awake at T-100. The analgesic score stayed highest (absence of PWR) from T-1 to T-45 (Table 1). HR and PE CO 2 did not show significant differences over time (p = 0.1 and 0.5, respectively). Respiratory frequency showed a significant decrease over time (p = 0.04), and was 70% lower than baseline at T-40 and T-45. SpO 2 showed a progressive decrease from T-0 to T- 15 (p = 0.001), increasing significantly over time (p < ) after O 2 supplementation. Blood-gas analysis at T-20 confirmed a hypoxemic state (PaO mmhg; kpa) accompanied by hypercapnia (PaCO mmhg; kpa) but not by acidosis (ph ). Arterial blood pressure progressively decreased over time (p = 0.03), with mean MAP values dropping from mmhg at T-1 to 47 9 mmhg at T- 45. Rectal temperature showed a biphasic trend, rising from at T-0 to C att Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

4 Table 1 Trends in physiological parameters over time in eight rabbits who received a combination of dexmedetomidine (DEX) (0.1 mg kg 1 ), midazolam (MID) (2 mg kg 1 ) and butorphanol (BUT) (0.4 mg kg 1 ) by the transnasal route Time points T0 T1 T5 T10 T15 T20 T25 T30 T35 T40 T45 HR (minute 1 ) f R (minute 1 ) 100 (60 150) 42 (25 109) 51 (18 100) 48 (10 94) 59 (10 123) 68 (15 107) 55 (19 117) 58 (19 77) 48 (17 72) 33 (23 66)* 38 (32 65)* T ( C) * * SpO2 (%) 96 (95 99) 92 (89 94) 90 (80 94)* 88 (83 92)* 87 (80 93)* 87 (86 90)* 90 (89 96) 92 (89 96) 92 (90 98) 94 (93 98) 96 (95 100) PE CO 2 (mmhg) 34 (16 60) 29 (19 61) 31 (14 60) 27 (20 62) 32 (25 46) 31 (18 44) 36 (16 44) 40 (20 46) 38 (27 55) 37 (31 53) PE CO2 (kpa) 4.5 ( ) 3.9 ( ) 4.1 ( ) 3.6 ( ) 4.3 ( ) 4.1 ( ) 4.8 ( ) 5.3 ( ) 5.0 ( ) 4.9 ( ) SAP (mmhg) 110 (80 216) 101 (72 121) 95 (60 117) 81 (57 102) 83 (58 97) 76 (61 86) 75 (66 93) 77 (69 85) 75 (60 103) 73 (61 92) MAP (mmhg) DAP (mmhg) Analgesia Score Sedation Score 2(0 2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (2) 2 (1 2) 2 (1 2) 11 (8 12) 12 (10 12) 12 (10 12) 11 (10 12) 11 (11 12) 11 (9 12) 10 (7 12) 10 (8 12) 9 (7 12) 8 (5 12) T0 = preinjection, baseline; T1 = loss of righting reflex; T- 10, 20, 25, 30, 35, 40 and 45 = minutes after T1. Parametric data are expressed as the mean SD, whereas non-parametric data are expressed as median (range). HR, heart rate; fr, respiratory frequency; T, temperature; SpO2, peripheral haemoglobin oxygen saturation; PE CO2, end-tidal carbon dioxide; SAP, systolic arterial pressure; MAP, mean arterial pressure; DAP, diastolic arterial pressure. *Significantly different form T0 (p < 0.05) Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

5 10 and then steadily decreasing to Cat T-45 (p < ). All animals recovered spontaneously and uneventfully within 1 hour and 40 minutes. Discussion Nasal catheterization is an easily performed and well-tolerated procedure in rabbits under gentle physical restraint. Some subjects noticeably swallowed at the time of drug administration; however, the short latency of drug action ( minute) after TN administration of our DMB combination rules out drug absorption by the gastrointestinal tract, supporting the efficacy of the transnasal route in rabbits. Respiratory frequency was severely reduced, although hypoxemia was lessened by O 2 supplementation. The observed respiratory depression can be ascribed to all three components of our protocol. The expected depressant effects of BUT added up to those of MID, which has been shown to contribute to respiratory depression in rabbits (Henke et al. 2005; Schroeder & Smith 2011; Hedenqvist et al. 2013), even after intranasal administration (Robertson & Eberhart 1994). Furthermore, DEX has also been reported to depress significantly ventilation, as well as the ventilatory response to CO 2 in healthy rabbits in a dose-dependent fashion (Nishida et al. 2002). Alpha-2 agonists are known to cause marked bradycardia and hypotension in rabbits (Hedenqvist et al. 2002; Henke et al. 2005). The consistent arterial pressure drop after administration of 0.1 mg kg 1 DEX, left untreated in our subjects, is in agreement with the findings of Henke et al. (2005), who used a similar drug combination in rabbits. Our DMB combination has the advantage of producing prompt and deep sedation along with profound analgesia in rabbits as shown by the absence of the PWR (Table 1), which has been identified as surgical anaesthesia and analgesia by other authors (Hedenqvist et al. 2002). Sedation onset was very short ( minutes) compared with that achieved with similar drug combinations given by injection (Hedenqvist et al. 2002; Henke et al. 2005). Immediate immobility and profound analgesia lasting up to 45 minutes are adequate for performing diagnostic procedures and minor surgery. In our study, all subjects woke spontaneously within 100 minutes of DMB administration without the need to antagonize any of the components. Reversing the effects of single components according to individual need can adjust the length and the depth of sedation to the requisites of different clinical procedures hastening recovery. As in most published sedative protocols for rabbits, including intranasal ones (Robertson & Eberhart 1994; Henke et al. 2005), our DMB combination requires O 2 supplementation and careful monitoring of vital parameters. In conclusion, the TN administration of our DMB combination is a powerful means for chemical restraint of tame and wild lagomorphs, adequate for 45 minutes long, painful procedures. Such a combination should be used cautiously in patients affected by cardiovascular or respiratory pathologies. References Eagleson JS, Platt SR, Elder Strong DL et al. (2012) Bioavailability of a novel midazolam gel after intranasal administration in dogs. Am J Vet Res 73, 6. Hedenqvist P, Orr HE, Roughan HE et al. (2002) Anaesthesia with ketamine/medetomidine in the rabbit: influence of route of administration and the effect of combination with Butorphanol. Vet Anaesth Analg 29, Hedenqvist P, Edner A, Fahlman A et al. (2013) Continuous intravenous anaesthesia with sufentanil and midazolam in medetomidine premedicated New Zealand White rabbits. BMC Vet Res 9, 21. Henke J, Astner S, Brill T et al. (2005) Comparative study of three intramuscular anaesthetic combinations (medetomidine/ketamine, medetomidine/fentanyl/ midazolam and xylazine/ketamine) in rabbits. Vet Anaesth Analg 32, Nishida T, Nishimura M, Kagawa K et al. (2002) The effects of dexmedetomidine on the ventilatory response to hypercapnia in rabbits. Intensive Care Med 28, Portnoy LG, Hustead DR (1992) Pharmacokinetics of butorphanol tartrate in rabbits. Am J Vet Res 53, Raekallio M, Ansah OB, Kuusela E et al. (2002) Some factors influencing the level of clinical sedation induced by medetomidine in rabbits. J Vet Pharmacol Ther 25, Robertson SA, Eberhart S (1994) Efficacy of the intranasal route for administration of anesthetic agents to adult rabbits. Lab Anim Sci 44, Schnellbacher RW, Hernandez SM, Tuberville TD et al. (2012) The efficacy of intranasal administration of dexmedetomidine and ketamine to Yellow-Bellied Sliders (Trachemys scripta scripta). J Herpetol Med Surg 22, Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

6 Schroeder CA, Smith LJ (2011) Respiratory rates and arterial blood-gas tensions in healthy rabbits given buprenorphine, butorphanol, midazolam, or their combinations. J Am Assoc Lab Anim Sci 50, Received 3 June 2014; accepted 3 February Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Sedation modified numeric rating scale for rabbits by Raekallio et al. (2002) Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesia and Analgesia, 43,

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