Some research procedures require the use of more specialized anaesthetic techniques. The use of NMB drugs may be needed, particularly for cardiothoracic and neurophysiological procedures. Use of these agents is challenging, and requires careful monitoring of anaesthetic depth. Their use will also require use of controlled ventilation and an understanding of the function of mechanical ventilators and the various types of apparatus that are available. Mechanical ventilation may also be needed during long-term anaesthesia, another challenging technique. Maintaining anaesthesia for prolonged periods can be achieved using a variety of ways. The advantages and disadvantages of the different techniques in relation to the scientific aims of the procedure, the experience of the staff, the welfare and well-being of the animals involved must be carefully considered.
Some groups of animals also provide particular challenges, for example anaesthesia of pregnant animals, fetuses and neonates. Finally, this chapter briefly outlines some of the particular issues relating to anaesthesia for non-invasive imaging.
NMB drugs produce paralysis of the skeletal muscles. They may be used either to aid stable mechanical ventilation by blocking spontaneous respiratory movements or, more frequently, to provide more suitable conditions for surgery. If skeletal muscle tone is eliminated by using a NMB agent, exposure of a surgical site can be achieved more easily and with less trauma to the surrounding tissues. NMB drugs are also used in neurophysiological and other studies, to enable very light planes of anaesthesia to be maintained. Under these conditions, if a relaxant had not been administered, spontaneous muscle movements could occur which would interfere with data collection.
The NMB drugs in common clinical use are classified as either depolarizing or non-depolarizing agents (Bowman, 2006). Depolarizing agents, such as suxamethonium and decamethonium, act similarly to the normal transmitter at the neuromuscular junction, acetylcholine. They bind to muscle receptors and trigger a muscle contraction but then produce a persistent depolarization, so preventing further muscle contractions. When drugs that act in this way are administered to an animal, generalized disorganized muscle twitches (fasciculations) are produced before complete skeletal muscle paralysis.
Non-depolarizing, or competitive blocking agents do not cause a muscle contraction before producing paralysis. Drugs in this group include tubocurarine, gallamine, pancuronium, alcuronium and vecuronium (Table 4.1). Since these agents act by competing with acetylcholine for receptor sites at the neuromuscular junction, their action can be reversed by increasing the local concentration of acetylcholine. This can be achieved by administering drugs such as neostigmine that block the activity of the enzymes which normally break down acetylcholine.
Table 4.1 Dose Rates for Neuromuscular Blocking Agents (mg/kg, by Intravenous Injection).
NMB agents must be used with great care, since their administration prevents all movements in response to pain. It would be possible, but obviously inhumane, to carry out a surgical procedure on an animal which had been paralysed but was still fully conscious. It is for this reason that the use of NMB drugs in experimental animals is subjected to careful control in many countries, for example special permission is required to use these agents in the UK, and Institutional Animal Care and Use Committee review is required in the USA. NMB agents are nevertheless extremely useful adjuncts to anaesthesia and enable, for example the use of balanced anaesthetic regimens such as an opioid, an hypnotic and a muscle relaxant to provide stable surgical anaesthesia. Dose rates of a number of different NMB agents are given in Table 4.1.
If NMB drugs are used, other methods of assessing the depth of anaesthesia must be adopted. As a preliminary step, the proposed anaesthetic technique, excluding the NMB drug, should be administered to an animal of the same species and the proposed surgical procedure carried out. This will establish that the degree of analgesia and unconsciousness will be sufficient to allow surgery to be carried out humanely. Since considerable individual variation in response to anaesthesia occurs and some inadvertent alteration in the technique can arise, for example due to equipment malfunction, it is also necessary to provide an independent assessment of the depth of anaesthesia. Several indicators of anaesthetic depth are of use. Despite muscle paralysis, twitching of muscles may occur in response to a major surgical stimulus and this indicates that the depth of anaesthesia is inadequate. In humans, pupillary size may alter in response to surgical stimulation, but this sign is of little value in most animals, particularly if atropine has been included in the pre-anaesthetic medication.
Changes in blood pressure and heart rate are the most widely used indicators of adequacy of the depth of anaesthesia. Dramatic changes in heart rate or blood pressure are believed to indicate a depth of anaesthesia insufficient for the surgical procedures that are being undertaken . It has been suggested that increases in heart rate and blood pressure by 10–20% indicate the need for additional anaesthesia. However, many anaesthetics do not block these autonomic responses and 10–20% increases in heart rate can be seen in animals that have not received NMB drugs, and yet these animals show no movement in association with the stimulus. If inadequately anaesthetized, most animals respond to surgical stimuli with a rise in blood pressure, but some animals may show a fall in pressure. So despite their widespread acceptance, these parameters may not always be reliable indicators of adequate anaesthesia (Whelan and Flecknell, 1992).
An alternative method of monitoring the depth of anaesthesia is to use the EEG. Although this requires specialist equipment and expert interpretation, these may be available, especially if neurosurgical or neurophysiological studies are being carried out. Simple changes in the unprocessed EEG, such as onset of burst suppression, can be useful when using some anaesthetic agents, for example halothane. Various derived measures, for example total power and spectral edge frequency have also been used to assess depth of anaesthesia (Murrell and Johnson, 2006; Otto, 2008); however, these measures generally cannot be used easily with balanced anaesthetic techniques which involve simultaneous use of hypnotics and analgesics. These same difficulties occur in human subjects, and great efforts have been made to develop monitoring devices that can measure loss of consciousness. The most recently developed have been bispectral index (BIS) monitors (Appadu and Vaidya, 2008), and these are now widely used in human patients, particularly in North America. Initial studies suggest that the instrument may also be of value in animals (Antognini et al., 2000; Lamont et al., 2004; Martin-Cancho et al., 2006). The attraction of this monitor is that it provides a single number as an index of consciousness, or depth of anaesthesia. The disadvantage is that, like the EEG from which it is derived, it is primarily intended to assess the degree of loss of consciousness, and is most predictive when a single anaesthetic agent is used. It is less reliable when using balanced anaesthesia. A further drawback is that it is designed for use in human beings, and the mathematical processing used to create the ‘index’ has been derived from measures made in large numbers of human subjects. Despite these problems, BIS monitors may be of use, particularly for long-term anaesthetic procedures with neuromuscular blockade, but extensive validation for each specific protocol will be needed before these monitors can be relied upon. It is also apparent that BIS values may vary between species at equivalent anaesthetic depths (Lamont et al., 2004).
Given the difficulties of monitoring the level of consciousness in paralysed animals, a more simple approach is to allow the action of the muscle relaxant to subside periodically. The animal will then be capable of responding to painful stimuli with voluntary movements. The degree of neuromuscular blockade can be monitored using a peripheral nerve stimulator. This device delivers a small electrical stimulus, either using skin electrodes or needle electrodes, to a peripheral nerve supplying muscle. In a non-paralysed animal the stimulation causes a muscle twitch. Allowing the actions of the muscle relaxant to subside will not always be practicable, especially during prolonged neurophysiological studies, however it is almost always feasible to delay administration of the relaxant until after the start of the surgical procedure. This allows an initial assessment of the adequacy of the depth of anaesthesia to be obtained. It also avoids difficulties in interpreting changes in heart rate and blood pressure that can occur as a side-effect of administration of some muscle relaxants (Rowlee, 1999; Appadu and Vaidya, 2008).
Decisions as to what constitutes an appropriate depth of anaesthesia, especially in paralysed animals, remains controversial. It has been suggested that very much lighter planes of anaesthesia should be used routinely (Antognini et al., 2005), but this approach does not take account of our current poor knowledge of indicators of consciousness in animals. A more conservative approach (e.g. Drummond et al., 1996) is recommended by most regulatory authorities, scientific journals and is adopted at the author’s own institution.
Many anaesthetic agents depress respiration and this can lead to the production of hypercapnia, hypoxia and acidosis. To maintain blood carbon dioxide and oxygen concentrations within normal levels, it is often necessary to assist ventilation. If the thoracic cavity is opened, the normal mechanisms of lung inflation are disrupted and it is usually necessary to ventilate the animal’s lungs artificially. It is not necessary to use a mechanical ventilator provided a suitable anaesthetic breathing system is in use (see Chapter 3), but using a ventilator will often be more convenient than manually assisting ventilation. A mechanical ventilator will often allow the precise control of the duration of inspiration and expiration, the volume of gas delivered to the lungs and the pressure reached in the airway during inspiration. It is not necessary to administer a NMB agent (muscle relaxant, curare-like drug) in order to carry out artificial ventilation but, unless the animal is deeply anaesthetized or is hyperventilated to produce hypocapnia, spontaneous respiratory movements may occur and these may interfere with ventilation and surgery.
Ventilators for use with animals may have either been specifically designed for these species, or may be adapted from their original use as ventilators for human subjects (Table 4.2). Ventilators are designed to achieve controlled ventilation of the animal’s lungs by means of the application of intermittent positive pressure to the airway. This may be achieved either by delivering gas directly to the anaesthetic breathing system or, indirectly, by compressing a rebreathing bag or bellows, which in turn delivers gas to the animal.
Table 4.2 Suggested Ventilation Rates for Laboratory Animals.
Tidal volumes of 10ml/kg are normally required. Whenever possible the adequacy of ventilation should be assessed by monitoring the end-tidal carbon dioxide concentration or by arterial blood gas analysis.
A variety of techniques have been devised to control the delivery of gas to the patient and to determine the patterns of gas flow and gas pressure that occur during ventilation. It might be thought that all that was required of a ventilator was to deliver the required volume of gas to the lungs at a predetermined rate. However, since the characteristics of the patient’s lungs, changes in airway resistance and leaks in the anaesthetic breathing system can all influence the volume of gas delivered, different techniques for terminating inspiration have been devised.
There are basically only two ways in which gas can be delivered during inspiration. The ventilator may deliver gas at a set pressure pattern: the pressure is determined by the machine, but the patient’s airway characteristics will influence the volume of gas which is delivered; this is because the pressure reached in the airway depends upon the resistance to flow provided by the patient’s lungs. If the ventilator is set to achieve a predetermined pressure, it will be reached earlier, and less gas will be delivered, if the patient’s lungs provide a higher resistance to flow.
In contrast, a ventilator may be set to produce a fixed flow pattern, which will be uninfluenced by the patient’s lung characteristics. Under these circumstances, the flow of gas will be constant but the pressure that develops in the airway will vary depending upon the patient’s lung characteristics.
It is important to understand how these two types of ventilators, termed ‘pressure generators’ and ‘flow generators’ respectively, are switched or cycled from inspiration to expiration. This can be achieved in several different ways, but the most frequently used method in animal ventilators is time cycling. Here, the change to expiration occurs after a preset time and is uninfluenced by changes in the patient’s lungs. If a time-cycled ventilator is used, the pressure developed in the lungs, the gas flow and the volume delivered can all vary. The actual values of these variables will depend both upon the characteristics of the patient and upon whether the ventilator is a pressure or flow generator. If the power of the ventilator is very great relative to the resistance of the patient’s lungs then, although time-cycled, the ventilator may in fact deliver a preset volume during inspiration.
An alternative to time cycling is to determine the volume of gas that should be delivered during inspiration based on the animal’s estimated tidal volume and change from inspiration to expiration when this volume has been delivered. In contrast, the changeover may be triggered not when a fixed volume of gas has been delivered, but when a predetermined airway pressure has been reached.
Once the lungs have been inflated and expiration begins, some mechanism must be used to trigger inspiration. In practice, only two techniques are used: either the changeover can occur after a fixed time or after airway pressure falls to a preset level.
The apparent complications introduced by the mechanics of ventilator design do have real effects on the patient. For example, if a fixed tidal volume is delivered, there will be no compensation for leaks in the anaesthetic breathing system so, if any leaks are present, there will be a fall in the volume of gas actually delivered to the lungs. A ventilator set to deliver gas until a preset pressure is achieved will compensate for leaks in the anaesthetic breathing system, but an increase in the animal’s airway resistance will result in a fall in the tidal volume delivered to the lungs. Ventilators that deliver gas at a fixed flow with a high generating pressure and that are either time or volume cycled, are unaffected by changes in the patient’s lungs, but they may produce excessive airway pressures.
In selecting a ventilator for use in laboratory animals, the most important factor to be considered is the ability to ventilate a wide range of animal species. It should be emphasized that the successful delivery of small tidal volumes (e.g. less than 50ml) often requires a leak-proof anaesthetic breathing system and minimal compliance of breathing system components such as connecting tubing. Additional features that may be needed are the ability to apply PEEP and a facility for humidification of gases. It is also important to select a machine that is simple to use and is reliable and easy to maintain. The author’s personal preference for a suitable multi-purpose ventilator is the Merlin ventilator (Vetronic Services, Appendix 4) (Fig. 4.1) which can deliver a wide range of tidal volumes ranging from 1 to 800ml.
An important practical consideration is that some ventilators require a source of compressed gas to provide the driving power for the ventilator. If a piped gas supply is available, then this does not represent a particular problem. If small gas cylinders are used to drive the ventilator, then large numbers may be required during a prolonged anaesthetic, even when used on a relatively small animal. Since the driving gas does not reach the animal’s lungs, a compressor delivering medical air is one possible solution. Alternatively a large cylinder of compressed air can be provided as the driving gas. If none of these solutions are thought practicable, then a mechanically driven ventilator is required. Most of these ventilators are designed to ventilate the animal either with room air or with gas provided from an anaesthetic machine. If gas is supplied from an anaesthetic machine, then it is important that a pressure relief valve is incorporated into the breathing system between the fresh gas inflow and the ventilator to prevent over-inflation of the animal’s lungs. Most ventilators designed for clinical use incorporate this highly desirable feature.
Separate controls for inspiratory and expiratory time may not be provided; some ventilators have only a control for the inspiratory time, and one for the inspiratory:expiratory (I:E) ratio. During IPPV, the heart and large veins in the thorax are compressed during inspiration, in contrast to the negative pressure that develops in the thorax during inspiration with spontaneous ventilation. The positive pressure produced during IPPV can reduce cardiac performance and cause a fall in blood pressure. To reduce this effect, inspiration should be completed in as short a period as possible, but must not be too rapid as this could result in high airway pressure. Conventionally, I:E ratios are set to be 1:2, but ratios of 1:3 and 1:4 will often cause less cardiac depression, while maintaining inflation pressures below 20cm water.
After setting the rate and tidal volumes, and I:E ratio (if possible), set the maximum inspiratory pressure – this should be less than 15cm water for small animals and should not exceed 25cm water in most circumstances.
To monitor inflation pressures, if the ventilator is not equipped with a pressure monitor, place a needle in the inspiratory side of the anaesthetic breathing system and attach it to a pressure transducer. Besides checking that excessive pressures do not develop, by setting appropriate limits on the pressure monitor, it can act as an alert should the animal become disconnected from the breathing system or the ventilator malfunction.
When the chest is open, the lungs collapse completely, and to prevent this many ventilators allow a positive pressure to be maintained at the end of expiration (PEEP). Only very low pressures, ranging 1–5cm of water, are normally required for small animals. PEEP can be applied either via a specific feature on some ventilators, or by attaching a PEEP valve onto the ventilator. In some models of rodent ventilator (e.g. the Harvard volume cycled model, Fig. 4.2), PEEP can be achieved simply by immersing the end of the tubing from the expiratory gas port into a few centimetres of water.
If a muscle relaxant is not being used to prevent spontaneous respiratory movements, then these can occur and interfere with the breathing cycles produced by the ventilator. One technique that can often reduce or eliminate these spontaneous movements is to increase the respiratory rate by approximately 50%, and slightly over-ventilate the animal for a few minutes. The respiratory rate can then be reduced slowly, and in many animals any spontaneous movements will remain suppressed, or will be occurring in synchrony with the ventilator.
Setting up and managing IPPV can seem daunting, but it is particularly useful in long anaesthetics (>1hour). Many veterinary or medically qualified anaesthetists should be able to provide expert advice. Once some simple protocols have been established, IPPV should be a relatively easy technique to master. Two recommended articles that provide a straightforward account of IPPV for veterinary practice are Dugdale (2007a) and (2007b).
When animals are anaesthetized for only a short period, their ability to withstand numerous disruptions to their normal physiology will often enable them to survive even very poor anaesthetic techniques. As the period of anaesthesia is extended, the adverse effects caused by poor technique become increasingly important. Similarly, the undesirable side-effects of many anaesthetic drugs become more apparent and a considerably higher standard of intra-operative care becomes necessary. Long-term anaesthesia is of course an arbitrary term, but here it is used to describe anaesthesia lasting longer than 60minutes.
There is little practical difference between anaesthesia from which the animal will be allowed to recover and that in which the animal will be killed at the end of the procedure. Prolonged, non-recovery anaesthesia, often undertaken to enable the study of physiological mechanisms or drug metabolism, usually requires stable anaesthesia with minimal depression of the various body systems. However, since recovery is not required, cumulative effects of drugs become less important, provided physiological stability can be maintained.
It might be thought that the simplest method of prolonging anaesthesia would be to give repeated doses of an injectable anaesthetic. Two problems arise if this approach is adopted. Giving intermittent doses of the drug will cause the depth of anaesthesia to vary considerably, although this can be overcome by administering it as a continuous infusion so that steady plasma concentrations of the anaesthetic are maintained. A second problem arises because of the pharmacokinetics of the anaesthetic. Following an initial injection of, for example a barbiturate, the blood concentration of the drug rises rapidly and the concentration in tissues with high relative blood flows, such as the brain, also increases rapidly. Redistribution of the drug to other body tissues then follows, with equilibration with body fat occurring most slowly. As this redistribution occurs, the concentration of drug in the brain falls. Recovery from the anaesthetic effects of the drug is primarily due to this redistribution, rather than to drug metabolism or excretion. If a second dose of anaesthetic is given, redistribution occurs more slowly, since the body tissues already contain some of the drug, and the duration of anaesthesia is prolonged. Repeated doses will have progressively greater effects. In addition to extending the duration of surgical anaesthesia, the sleeping time following anaesthesia is also very prolonged. If the animal does eventually wake up, the residual effects of the drug may persist for 24–48hours. For this reason, repeated incremental doses of drugs such as the barbiturates are not an ideal way of prolonging anaesthesia. The cumulative effects of different types of anaesthetic do vary considerably and some, such as alphaxalone/alphadolone, alphaxalone and propofol, are rapidly metabolized following their administration. These drugs can be used to produce prolonged periods of anaesthesia without causing greatly extended recovery times (Coetzee, 2005).
Whichever injectable anaesthetic is used, it is preferable to administer incremental doses of the drug by the intravenous route, so that its effects on the depth of anaesthesia will be seen rapidly and be readily adjusted. Administration by other routes is possible with some drugs, but the depth of anaesthesia will vary less predictably.
Recovery following repeated doses of barbiturates is very prolonged, so the use of these drugs for procedures from which the animal is expected to recover consciousness is not recommended. Incremental doses of barbiturates can be used for non-recovery experiments, but the hypotension and respiratory depression that may result can cause serious problems. In addition, the depth of anaesthesia will vary considerably and may be insufficient to allow surgical procedures to be undertaken in rodents and rabbits. In larger species, continuous infusion of pentobarbital or repeated administration can be used successfully for long-term anaesthesia, but recovery is very prolonged and often associated with long periods of involuntary excitement and ataxia. The drug is therefore best reserved for use in non-recovery procedures.