EEG traces from joeys younger than about 100 days were nearly completely isoelectric (Table 2.2), as judged by relative EEG power of the EEG power spectrum compared to non-isoelectric EEG traces. Traces from older joeys also showed isoelectric periods interspersed between EEG epochs. Isoelectric periods were of 300msec or longer duration during which the amplitude of the EEG was at least 4-5 times lower than that of the remaining trace. This was assessed visually (see Figure 2.1). The EEG recordings over the first 3 minutes at a stable endtidal halothane concentration of 1% (baseline recording), over the first 3 minutes immediately after toe clamping and over the first 3 minutes at stable endtidal halothane concentrations of 1.2% and 1.4%, were used for examining isoelectric periods. The proportion of time occupied by isoelectric periods (%) and the total number and mean duration of these periods were recorded for all animals for each of these defined periods (Table 2.2).
program (Spectral Analyser, CB Johnson, Massey University, New Zealand, 2006), to calculate the median frequency (F50), spectral edge frequency (F95) and total power (Ptot) for consecutive 1-second segments as well as the power for frequencies of 1- 30Hz. Samples of continuous EEG of a duration of 20 seconds each were obtained from the beginning of baseline (1.0% endtidal halothane), during clamping, immediately after clamping and at the beginning of stable endtidal halothane concentrations of 1.2% and 1.4%. Where short isoelectric periods or artefacts were present (<5sec), calculated spectral data were substituted by the preceding value calculated in order to avoid invalid data (i.e. non-stationary data). In five joeys, isoelectric periods were too common to allow this approach. Therefore, five periods of the EEG traces (>3000msec) were selected at equivalent recording periods as those described above, subjected to FFT analysis and the resultant values were combined for each recording period (i.e. baseline, clamping, post-clamping, 1.2% and 1.4% endtidal halothane) for each animal.
Total power and EEG power (log power) of the individual frequency bands have been reported here as arbitrary units. This was done as the set-up of the present study did not allow for the amplification effect of the signal to be taken into consideration. Hence no units could be ascribed. As the same system was used throughout the study and no changes were made to the recording apparatus, all values were recorded on the same scale and can be directly compared with each other without the use of defined units.
Figure 2.1: EEG traces of joeys approximately 94 days, 124 days, 145 days, 173 days, 198 days and 261 days of in-pouch age (from top to bottom), showing an isoelectric EEG at 94 days and isoelectric epochs for joeys at 124 and 145 days of in-pouch age. Note the different scale for the two oldest joeys – the first scale shown relates to the four traces above it and the second to the two traces above it.
Table 2.2: Percentage of time occupied by isoelectric EEG periods (%), average duration of isoelectric periods (msec) and number of isoelectric periods present (N) during 3 minutes of EEG recordings during baseline, post clamping and at endtidal halothane concentrations of 1.2% and 1.4%. Joeys that could not be intubated (n=3) and hence only had a 5-minute EEG record taken are not included in this table, but had an isoelectric EEG throughout the 5 minutes (100%).
1.0% After clamping 1.2% 1.4%
Age
(days) % msec N % msec N % msec N % msec N
94 99 35426 5 99 35220 5 100 180000 1 100 89620 2 104 94 8033 20 92 6284 25 96 11906 14 100 179279 1 114 97 21908 8 91 7131 23 96 13236 13 99 59520 3 118 78 3804 37 78 3807 37 77 5990 23 94 15455 11 124 55 2010 49 52 2078 45 57 2314 44 68 3140 39 127 87 5421 29 80 4380 33 78 3803 37 81 4675 31 142 5 657 13 7 851 15 11 1045 19 22 1222 32 145 13 1072 22 11 1015 20 16 909 32 29 1408 37 164 0 0 0 2 377 9 3 719 8 173 0 0 0 0 0 0 0 0 0 1 350 1 174 2 663 6 2 616 7 6 925 11 9 860 19 181 0 0 0 0 0 0 1 595 4 4 651 10 187 0 0 0 1 356 5 3 449 10 187 0 0 0 1 350 1 1 440 1 4 635 11 189 0 0 0 0 0 0 0 0 0 0 0 0 196 0 0 0 0 0 0 0 0 0 0 0 0 198 0 0 0 0 0 0 0 0 0 1 345 2 215 0 0 0 0 0 0 1 665 2 1 665 2 227 0 0 0 0 0 0 0 0 0 0 0 0 231 0 0 0 0 0 0 0 0 0 0 0 0 238 0 0 0 0 0 0 0 0 0 0 0 0 261 0 0 0 0 0 0 0 0 0 0 0 0
2) Joeys not anaesthetised during EEG recordings
The EEG traces of the four joeys that were not anaesthetised during EEG recordings were assessed for the presence of isoelectric periods (see definition above). Spectral analysis was then performed as above, using the specialised Fast Fourier Transform (FFT) program.
It is commonly known that sevoflurane is a short-acting anaesthetic due to its low solubility (Eger & Johnson, 1987; Strum & Eger, 1987). In addition, due to the size of wallaby joeys compared to adult wallabies we anticipated that recovery would be even faster in the joeys, as small animals have greater ventilation rates and cardiac output (Eger & Johnson, 1987). Although we do not know the sevoflurane recovery times for tammar wallabies, we used EEG traces from 10 minutes after termination of anaesthesia exposure onwards for FFT analysis. Residual anaesthetic concentrations that may have been present in the joeys at this time were not considered to have been sufficiently high to significantly affect spectral data. This is supported by a study where 3-month-old rats were reported to right themselves after about six minutes and to pass a Rotarod test on average after 15 minutes following two hours of sevoflurane anaesthesia at 1.6 MAC (Eger & Johnson, 1987).
The EEG traces of the present study were inspected for movement artefact and five non- contaminated 30-second EEG periods were chosen for FFT analysis from 10 minutes after the EEG recordings had commenced (i.e.10 minutes after anaesthetic exposure had been terminated).
As joeys were covered with a cardboard box to reduce light levels, we were not able to record their behaviour. Therefore, it was not possible to determine whether the animals were aroused or asleep during the recording period. Although movement artefact is present in the EEG traces and would suggest an aroused joey, this was not considered to be a reliable indicator of arousal/sleep state as joeys may have been aroused without causing movement artefact.
high frequency waves (LVHF, awake-like) and apparent high voltage low frequency waves (HVLF, sleep-like/quiet awake) were used for the purpose of the present study. The joey aged 155 days did not show HVLF EEG characteristics after recovery and hence only LVHF EEG data are available for this animal. The EEGs of the other three joeys did show both EEG characteristics, but movement artefact during the putative awake-like periods was common preventing meaningful FFT analyses. Thus, the data presented relate to HVLF EEGs of joeys aged 137, 189 and 193 days and awake-like EEG periods of the joey aged 155 days. However, in the absence of direct behavioural observations and the potential to correlate them with EEG traces, small movement artefact might have been present during the selected periods and not interpreted as such.
Statistical analysis
Statistical analysis was performed in SPSS 11.0 for MAC OSX (SPSS Inc., USA) and Prism 5 for MAC OSX (GraphPad Software, Inc., USA). Data were tested for normality using the Shapiro-Wilks statistic (if n<50) or the Kolmogorov-Smirnoff statistic (if n>50) and normal probability plots. The Levene’s test was used to test for homogeneity of the data where these were normally distributed. Data are presented as mean ± standard error of the mean (SEM). Differences were considered significant at p=0.05 or less. Degrees of freedom reported (e.g. df 1, 130) represent the number of groups and overall number of data points used in individual statistical tests.
1) Anaesthetised joeys