In the present study we created a series of anaesthetic situations that were monitored by clinical scores to assess anaesthetic depth and, in addition, the BIS. The results correspond to classical findings during anaesthesia monitoring and provide new original data on the isoflurane-sevoflurane comparison in an avian species as well as additional effects of two sedative and analgesic drugs. The main finding was that the BIS recorded a level of anaesthetic depth different from the one deducted from VAS monitoring alone.
Two constraints apply to the present study. It is known that invasive blood pressure monitoring provides more reliable results than the NIBP . Yet, even though this may make our blood pressure values not directly comparable to other studies, they are meaningful within our study, where the same method was used consistently measuring the same individuals repeatedly on different treatments. Another common monitoring measurement, the respiratory rate, could not be used to assess anaesthesia depth since the chickens were mechanical ventilated to maintain normocapnia. This was considered necessary because hypercapnia might have influenced cerebral blood flow and consequently could have made the BIS readings more variable .
As was observed in other avian species the mean MAC for sevoflurane (1.90 ± 0.26%) was higher than for isoflurane (1.15 ± 0.20%) . Typical reactions to the anaesthetic protocols were observed. As in humans , the level of hypnosis was not affected differently by the use of either gas. The indication of a trend for higher VAS on sevoflurane (Table 2), in particular when combined with other sedative and analgesic drugs (Fig. 1), suggests that additive effects may be less distinct in sevoflurane compared to isoflurane. Studies on medetomidine in the avian patient have been restricted to the evaluation of the sedative effects of this drug alone, or to assess the adequacy of anaesthetic protocols including not only inhalant anaesthetics and medetomidine, but also hypnotic drugs or benzodiazepines, and therefore little can be presumed about the character of the additive effect of medetomidine on the inhalation gases used in this study [25, 26]. The sparing effect of butorphanol on isoflurane and sevoflurane has been described in avian medicine, but not in the same species and in the same conditions as was done in this study, hence impairing direct comparison [23, 27]. Direct comparison of the anaesthesia using a combination of medetomidine with isoflurane or sevoflurane has been made in horses, hypothesizing that the differences observed between both combinations are a consequence of the lower partition coefficient of sevoflurane compared to isoflurane . Whilst the lower partition coefficient of sevoflurane may have an effect, it is unlikely to explain alone the additive effects of the combination in our study due to the long equilibration phase used to reach equipotent MAC multiples in each case. Larger sample sizes are evidently needed to clarify this issue.
The well-known reduction in systemic vascular resistance and arterial blood pressure produced by volatile anaesthetics corresponds to the decrease in NIBP and the compensating increase in HR at increasing gas concentrations . The different magnitude of NIBP-depressing effect of butorphanol when added to an isoflurane or a sevoflurane inhalation protocol has been previously observed in Guinea fowl (Numida meleagris) . Most importantly, the VAS decreased at increasing gas concentration as expected, and medetomidine but not butorphanol led to a distinct additional reduction in VAS. Butorphanol is frequently used in avian medicine to provide analgesia to the anaesthetic protocol due to the fact that birds are considered to possess mainly kappa receptors. The absence of lower VAS values when including butorphanol compared to the control group is noteworthy. This is unlikely an effect of opiate clearance, as a study in broiler chickens showed that butorphanol administered at a dose of 2 mg/kg IV maintained what is considered the minimal effective concentration for analgesia in mammals for a longer duration than the duration of the present study . A possible explanation for the lack of butorphanol effect may be the dose (1 mg/kg IV) used in the present study. Furthermore, heterogeneity of opiate receptor density and distribution in different avian species, and within the same species among individuals or, for example, among chicken lines have been described [19, 31,32,33,34]. The present study emphasizes that the use of opiates in general and specifically of butorphanol as an analgesic in birds needs further evaluation.
As described previously , BIS decreased at increasing gas concentrations, showed no correlation to HR, but correlated to blood pressure. The fact that also NIBP decreased with increasing MAC multiples needs to be considered when evaluating BIS values, since hypoperfusion seem to have a direct, negative effect on BIS . Inhalation anaesthesia based on sevoflurane or isoflurane in dogs has shown similar cerebral effects assessed by cerebral blood flow, EEG and cerebral metabolic depressant effect and burst suppression (SR) occurred at higher MAC multiples for both gases . In the present study, SR reached values higher than 40 at the deepest anaesthetics planes (equivalent to higher MAC multiples), as expected. Moreover, none of the anaesthetics and sedatives used produced a significant effect on this value, meaning that the brain activity suppression was independent of the anaesthetic protocol used and depended on the anaesthetic depth only. However, standard deviations of SR are very large, values > 0 were not only recorded during periods of deep anaesthetic planes at high MAC multiples and low BIS, but also at lower MAC multiples and at BIS values above 30. This has been previously observed in chickens and cats, suggesting that burst suppression is not a strict indicator of anaesthesia depth [15, 37].
Two findings of the present study indicate that the BIS records cortical activity that is not mirrored by the other methods of monitoring anaesthetic depth: First, the fact that the difference in BIS before and after stimulation does not remain constant, but increases with increasing MAC multiples, because the BISpost is not suppressed proportionately to the suppression of the BISpre values due to gas concentration. Second, the difference in the effect of medetomidine on VAS and BIS evaluation of anaesthetic depth.
It has been reported previously that the BIS is an individual characteristic . This was also confirmed in the present study, where the individual had a significant random effect when assessing BISpre and BISpost values (Table 2). This occurred despite the individual MAC for each animal had been determined first, to ensure comparability of the different levels of gas concentration [15, 39, 40]. In contrast, the difference between the BISpre and BISpost values showed no effect of individual, indicating that the general principle, i.e. the magnitude of change, is representative for the population. It is only the underlying baseline BIS value that is individual-specific. The BISpost response to the stimuli indicates that even though the level of anaesthesia is considered deep (as judged by VAS), these stimuli have an effect on the cortex and are registered in the EEG.
Similarly, the sedative effect of medetomidine led to a reduction of the VAS, but not to an additional effect on the BIS. This incongruence between BIS and VAS was easily explained by the fundaments of each anaesthesia monitoring technique, where BIS evaluates electrical activity in the frontal cortex, and VAS includes the neuromuscular response to reflex stimulation or the assessment of subcortical spinal inhibition. In dogs, the combination of medetomidine with isoflurane produced a decrease in BIS, which translates in a deeper level of hypnosis and cortical depression, compared to isoflurane administration alone . Isoflurane and sevoflurane are ethers that produce a reversible, dose-related CNS depression, inhibiting spinal and supraspinal areas and, at adequate concentrations, they produce anaesthesia. However, noxious stimulation may produce an increase in central activity directly, or indirectly by triggering hemodynamic changes through the sympathetic system . Medetomidine produces sedation, analgesia and muscle relaxation by binding to the several types of α2-adrenergic receptors throughout the body, including the cerebral cortex, and therefore able to produce spinal and supraspinal analgesia . The present study indicates that in this group of chickens medetomidine produced a less visible central depressing effect and therefore less noticeable effect on BIS, yet maintained its antinociceptive effect compared to the control groups and butorphanol, inhibiting the spinal pathway and therefore decreasing the reflex response to noxious stimulation.
BIS measurements of the present study indicate that anaesthetic scenarios exist where subcortical suppression occurs in parallel with cortical activity. This is similar to reports in humans, were hypothesis about the increase in BISpost might be related to noxious stimulation even with immobility . Moreover, in the same study, it is shown how lower BIS in the elderly patient compared to the young patient might indicate an overdose of the used anaesthetic and a lack of antinociception, producing unnecessary deep anaesthetic depth and allowing an improved titration of analgesia. The relevance of these findings is that there is less security that procedures undergone during what is considered an adequate anaesthetic plane based on haemodynamic and neuromuscular parameters do not leave traces in the awareness, and hence represent putatively stressful events for the patient. Studies attempting to link BIS to stress responses measured by catecholamine and cortisol and ACTH levels have been performed in humans with promising but as yet inconclusive results [43,44,45]. Meta-analyses linking BIS monitoring to awareness incidence in humans as yet failed to unequivocally reveal a superiority of this method, which calls for caution to uncritically link depth of anaesthesia monitoring to intraoperative awareness [46, 47].
For avian medicine the use of BIS has the potential to improve anaesthetic monitoring and as such to have an impact on welfare. BIS adds objective, continuous information regarding the CNS activity. This complements data regarding parameters such as HR and blood pressure, which are typically taken at set intervals, and reflex scores, which are subjective. The continuous measuring of BIS values allows the early detection of trends regarding anaesthetics depth and adds to the safety of anaesthetic procedures.