The rabbit is the third most commonly used species for experimental research in the European Union, and it is increasing in numbers 1 . It is also the third most common pet species anaesthetised in the United Kingdom 2 . Rabbits are high risk anaesthesia patients, with a mortality risk 14 times higher than in dogs 3 . Possible reasons for this are that rabbits are easily stressed prey animals, they are difficult to intubate endotracheally and react to mask induction with volatile anaesthetics by extended breath-holding 4 5 6 . Rabbits have a large abdominal cavity in relation to the thoracic cavity, with the results that the pressure from the intestinal mass may interfere with respiration in dorsal recumbency during anaesthesia. Furthermore, obesity is increasing in pet rabbits, which also adds to the problem. Respiratory infection with Pasteurella pneumotropica, which is common in pet rabbits, may cause a reduction of the hydrogen ion driven respiratory drive, adding to the anaesthetic risk 7 . Rabbits are also prone to develop substantial hypotension during anaesthesia 8 .

Anaesthesia of long-duration is often needed in experimental settings, as well as in clinical settings, as rabbit owners request more elaborate treatment for their pets. Inhalation anaesthesia is an option for long-duration anaesthesia although not all clinics or research institutions have access to the necessary facilities and equipment. Although significant research has been done to improve safety and reduce negative environmental impact, volatile anaesthetics may constitute an occupational health hazard and are toxic to the environment. Elaborate equipment is needed for waste gas scavenging, and there have been plans to phase out the use of volatile anaesthetics 9 .

In recent years continuous total intravenous anaesthesia (TIVA) has become popular in veterinary anaesthesia 10 . Advantages compared to volatile anaesthesia are: reduced cardiovascular depression, smooth recovery and reduced incidence of postoperative nausea 11 12 13 . The development of intravenous (IV) anaesthetics with a short half-life has made it possible to control duration and depth of anaesthesia in a manner similar to inhalation anaesthesia.

Several TIVA protocols have been evaluated in animals, including the use of opioids, benzodiazepines, ketamine and propofol. Of the few that have been described in rabbits, TIVA with either ketamine-xylazine 14 or propofol 15 16 does not provide a surgical plane of anaesthesia, and yet produces substantial negative effects on cardiovascular variables. TIVA with fentanyl in combination with either diazepam 17 or propofol 18 has only been examined in combination with neuro-muscular blocking agents, which may prevent evaluation of analgesic properties. Ketamine-fentanyl TIVA has been used in combination with local anaesthesia in rabbits for cranial surgery 19 , but the quality of anaesthesia was not reported.

High-dose opioid anaesthesia has been employed in experimental primate 20 and swine 21 surgery, with limited negative cardiovascular effects. Opioids inhibit sympathetic activity 22 and provide haemodynamic stability both during and after surgery. Sufentanil, which has been shown to inhibit sympathetic activity better than fentanyl in dogs 23 , was used successfully in combination with the benzodiazepine midazolam for major surgery in dogs that were cardiovascularly compromised, and was shown to provide good hemodynamic stability 24 . One benefit of using TIVA with sufentanil-midazolam is that its effects may be antagonized with commercially available antidotes.

For the above-mentioned reasons, sufentanil-midazolam TIVA may be useful in rabbit anaesthesia. Our aim was to evaluate the cardiorespiratory effects of a TIVA protocol with sufentanil-midazolam in medetomidine premedicated NZW rabbits. Variables of anaesthesia induction, maintenance and recovery are reported together with cardio-respiratory variables and concentrations of serum glucose, lactate, total protein, sufentanil and midazolam.

Data are presented as mean ± SD or median (range) and n = 8 unless indicated. Haematology results are presented in Table 1.

^a EPC erythrocyte particle concentration, Hb haemoglobin, LPK leucocyte particle concentration, EVF erythrocyte volume fraction, MCV mean corpuscular volume, MCHC mean corpuscular hemoglobin concentration.

The righting reflex was lost in 3 ± 1 min and the pedal withdrawal reflex in 5 ± 2 min after start of induction. Induction occurred without excitation or apnoea, and the number of intubation attempts ranged between 2 and 6. No apnoea or struggling was encountered during intubation. Time to intubation from start of induction was 9 ± 2 min (n = 7). Intubation failed in one rabbit and a close fitting mask (Galemed with connector, Kruuse svenska AB) was used instead. Induction doses for sufentanil and midazolam were 0.48 ± 0.11 μg/kg BW and 0.09 ± 0.02 mg/kg BW, respectively. Maintenance doses were 0.72 ± 0.15 μg/kg BW/h and 0.13 ± 0.03 mg/kg BW/h respectively, corresponding to a mean infusion rate of 0.29 ± 0.06 ml/kg BW/h. The infusion rate did not change statistically over 60 min of anaesthesia duration.

Maintenance of anaesthesia was characterized by muscle relaxation and loss of the pedal withdrawal reflex. Compared to values during sedation, the respiratory rate was reduced during the first 20 minutes of anaesthesia (Table 2). Two rabbits occasionally needed mechanical ventilation during maintenance due to apnoea. End-tidal CO₂ (ETCO₂) and arterial partial pressure of CO₂ (PaCO₂) were increased during most of the maintenance of anaesthesia (Figure 1). Blood pH decreased from 7.50 ± 0.00 to 7.28 ± 0.04 and serum lactate from 1.9 ± 0.6 to 0.7 ± 0.2 mmol/L (n = 7). The arterial partial pressure of O₂ (PaO₂) increased from 9.6 (8.4-11.1) to 38 (35–46) kPa whereas mean arterial pressure (MAP) decreased during anaesthesia (Table 2). Heart rate (HR) and cardiac output (CO, n = 6) increased immediately after induction and remained high during most of maintenance (Figure 2). No signs of arrhythmias were observed on the ECG.

Values are presented as median (range).

^avalues were recorded approximately 30 min after injection with medetomidine (0.1 mg/kg BW SC). ^bsignificantly different compared with values recorded in sedated rabbits. Friedman Repeated Measures ANOVA on ranks, multiple comparisons with Dunn’s method. *10 min after TIVA stop.

Rectal temperature dropped from 39.2°C (37.9–39.9) to 38.4°C (38.1–39.0) at 60 min. Blood glucose increased from 10.8 (6.6–14.0) to 16.0 (8.3-18.5) mmol/L (n = 7) and haematocrit from 23 ± 2 to 27 ± 1%. Sufentanil and midazolam serum levels did not change from 15 to 60 min during anaesthesia (Figure 3). Time from stop of TIVA infusion to extubation was 5 ± 3 min, and to recovery of the righting reflex 7 ± 6 min. Recovery was without excitation. Body weight was reduced by 3% 24 h after anaesthesia and restored 48 h later. No clinical abnormalities were seen during the week after anaesthesia.

TIVA with sufentanil-midazolam was evaluated in medetomidine pre-medicated NZW rabbits. Induction was without excitation, apnoea or muscle rigidity, providing good conditions for endotracheal intubation. Anaesthesia maintenance was characterised by absence of a pedal withdrawal reflex and muscle relaxation, although marked respiratory depression and hypotension were produced.

We successfully intubated the airways of 6 rabbits after 2–4 blind attempts. The conditions for intubation were good, as judged by lack of jaw muscle tone and reflex responses to intubation, and when an intubation attempt failed, the endotracheal tube ended up in the oesophagus. No laryngospasm, swelling of the larynx or bleeding was encountered. The difficulty of endotracheal intubation in rabbits has been described in numerous studies 25 26 27 . Rabbits have a narrow oral cavity which makes visual intubation difficult and successful blind intubation requires both practice and routine. Using a specially designed laryngoscope such as the Flecknell™ small animal laryngoscope (Alstoe Ltd. Animal Health, UK) may be helpful.

Apnoea during induction must be avoided, since it complicates blind intubation and rapidly leads to hypoxemia in rabbits, due to their high metabolic rate. To avoid apnoea, anaesthesia must be induced slowly. Midazolam alone can cause apnoea in humans if infused too quickly IV 28 . Slow induction also prevents muscle rigidity which occurs if sufentanil is administered too fast, which has been shown in pigs 21 and humans. In the currents study, catalepsy was not observed.

A similar IV drug combination (xylazine-alfentanyl-midazolam) has been evaluated in NZW rabbits 29 , although not as a continuous infusion. The effects on the respiratory and cardiovascular systems were similar to those seen in the present study, but muscle relaxation was poor and seizures were induced. In comparison, TIVA with sufentanil-midazolam seems to be a better alternative.

During development of the current protocol, premedication with fentanyl-fluanisone was abandoned due to occurrence of apnoea during induction of anaesthesia. This was not the case after premedication with medetomidine at 0.1 mg/kg BW. In combination with local anaesthesia cream, medetomidine sedation produced satisfactory conditions for placement of vessel catheters. During TIVA maintenance however, apnoea occurred occasionally in two rabbits.

Medetomidine is known for its adverse cardiovascular effects 30 , even in very low doses 31 . In human anaesthesiology, alpha-2-adrenergic agonists are increasingly used in low doses to improve cardiovascular stability and prevent tachycardia 32 .

Sufentanil-midazolam anaesthesia caused a marked decrease in respiratory rate and marked hypercapnia. The level of hypercapnia produced was similar to that produced by 2 MAC isoflurane 33 . Opioids are known respiratory depressants, and midazolam has been shown to slightly add to this depression in rabbits 34 . Despite marked respiratory depression, we chose to ventilate the rabbits minimally in order to evaluate the extent of respiratory depression. It is likely that the respiratory depression had an effect on other variables, such as the HR. However, experience shows that during short term anaesthesia, some degree of hypercapnia (6–7 kPa) is well tolerated in healthy cats and dogs 35 , and may even be beneficial since it stimulates respiration and increases blood flow to the brain 36 . The occurrence of apnoea during maintenance of anaesthesia necessitates the use of mechanical ventilation, which is the main disadvantage of the protocol since it requires successful endotracheal intubation as well as the knowledge and skills of using a ventilator.

The heart rate increased after induction with TIVA, although the values did not reach the levels reported in awake unrestrained rabbits 37 . A possible explanation for the increase is the sympathomimetic stimulation caused by endotracheal intubation 38 . Alpha-2-agonists are able to block cardiovascular responses caused by intubation and surgery in humans 39 and opioids can block the sympathetic response to surgery in dogs 23 24 . If intubation caused the increased HR in the present study, the medetomidine and sufentanil doses used must have been insufficient to prevent the response. The simultaneous hypercapnia and hypotension that developed may also have contributed to the increase in HR. Isoflurane increases heart rate by 20% because of depression of the vagal tone rather than an increase in the sympathetic tone 40 .

Rather than an increase in HR we had anticipated a decrease, since opioids are known to cause bradycardia through medullary vagal stimulation 41 . Sufentanil-medetomidine anaesthesia, albeit in a higher doses than in the present study and without the concurrent use of an alpha-2-adrenergic agonist, causes bradycardia in dogs 24 as well as humans 42 .

Marked hypotension developed during anaesthesia (MAP 46 mm Hg), yet all rabbits recovered uneventfully. A high prevalence of hypotension, defined as a MAP < 60 mm Hg, has been reported in rabbits anaesthetized with e.g. isoflurane, haltothane or ketamine-medetomidine 8 33 43 . Maintenance of anaesthesia with isoflurane in NZW rabbits will give rise to MAP in the range of 33–82 mmHg 8 . The pressure measured in the auricular artery has been shown to be approximately 8 mm Hg lower than in the carotid artery 38 . In the present study MAP in the sedated rabbits (~70 mmHg) was lower than reported in non-sedated unrestrained resting rabbits (~80 mmHg), 37 and similar to MAP in rabbits after IV administration of 10 μg/kg BW dexmedetomidine (~75 mmHg), 44 . Midazolam alone 45 or in combination with sufentanil 24 , has been shown to decrease vascular resistance and blood pressure in dogs, however MAP did not fall below 65 mmHg 24 . In humans, sufentanil-midazolam also decreases MAP (~75 mm Hg), 46 47 . In contrast to the present study, the human and dog studies included major surgery, which may have affected MAP. Uncorrected hypotension may contribute to the high mortality levels in rabbit anaesthesia 8 and cardiovascular support, which is routine in other animal species during anaesthesia, may possibly decrease mortality rates.

MAP and measurement of lactate concentrations 48 are indirect measures of tissue perfusion. The serum lactate levels in the present study indicate that tissue perfusion was adequate despite the low MAP. The levels were low before and during anaesthesia (0.7-1.9 mmol/L), similar to those seen in rabbits lightly anaesthetised with propofol (1.3 mmol/L), 49 and considerably lower compared to non-sedated, restrained rabbits (7.3 mmol/L), 50 . Increased lactate levels are a sign of tissue hypoxia and anaerobic metabolism and should be treated with therapy directed at improving tissue oxygen delivery. The acidosis that quickly developed after induction of anaesthesia was most likely of respiratory origin.

Tissue perfusion is likely to be compromised if vasoconstriction and reduced cardiac output are present. In the current study, however, CO increased after anaesthesia induction, which was likely a consequence of the increased HR and decreased MAP. Midazolam has been shown to increase cardiac output in dogs at doses as low as 1 mg/kg 45 .

CO was measured with the lithium-dilution technique, which has not yet been validated in rabbits. It has been validated in human babies with a similar BW (~2 kg) and found to be accurate in comparison to transpulmonary thermodilution 51 . It has also been validated in dogs 52 and cats 53 . The benefit of the technique is that a peripheral instead of a central vein may be used, which reduces the risk for complications. No adverse effects were seen due to the CO measurement in the present study. Values recorded in medetomidine sedated rabbits were similar to levels measured with a transit time Doppler in NZW rabbits sedated with dexmedetomidine 0.01 mg/kg BWIV 44 .

The glucose levels in pre-medicated rabbits were high (7–14 mmol/L) compared to those reported in non-sedated rabbits (4–8 mmol/L), 54 . A further increase in glucose was seen after anaesthesia induction, reaching a maximum of 8–18 mmol/L at approximately 50 min after medetomidine administration. Medetomidine is known to decrease insulin levels 55 and has been shown to increase blood glucose levels in rabbits 56 . Midazolam on the other hand, has been shown to decrease glucose levels during surgery in humans 57 and, furthermore opioids inhibit the entire sympathetic nervous system 22 . If the sufentanil-midazolam doses were not high enough to fully inhibit sympathetic activity, the stimulation caused by endotracheal intubation may have contributed to the increase in glucose levels.

Serum concentrations of sufentanil (0.60 ng/mL) and midazolam (140 ng/mL) remained constant throughout anaesthesia, with no tendency to accumulate. Plasma concentrations have been measured in humans undergoing heart surgery during sufentanil-midazolam anaesthesia 46 . Compared to the present study, the infusion level was higher for sufentanil and lower for midazolam and accordingly, the plasma levels higher for sufentanil (2–4 ng/mL) and lower for midazolam (75–100 ng/mL).

To further evaluate this protocol, experimental work needs to be undertaken in both healthy and sick rabbits during surgery and in comparison with inhalation anaesthesia.

TIVA with sufentanil-midazolam provided smooth induction and recovery of anaesthesia in rabbits, but with marked hypotension and respiratory depression, requiring mechanical ventilation. Despite the side effects, the protocol may still be an alternative to inhalation anaesthesia in situations where longer duration anaesthesia is needed and inhalation anaesthesia is not an option. The fact that endotracheal intubation and mechanical ventilation is necessary makes the protocol a less likely alternative for the general practitioner.

The study was approved by Uppsala, Sweden, Experimental Animal Ethics Committee (C368/9).

The authors declare that they have no competing interests.

AE, PH and MJW designed the study and all authors contributed to the acquisition of data. PH analysed the data and all authors contributed to interpretation of data, drafting and revision of the manuscript. All authors read and approved the final manuscript.