International Journal of Audiology 2014; 53: 250–258
Original Article
Influence of dose and duration of isoflurane anesthesia on the auditory brainstem response in the rat
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Eric C. Bielefeld Department of Speech and Hearing Science, The Ohio State University, Columbus, Ohio, USA
Abstract Objective: Isoflurane anesthesia can have significant effects on processing of sounds at the peripheral and central levels, manifesting in changes in auditory-evoked potentials. The current study tested whether duration of isoflurane anesthesia changes thresholds, amplitudes, and latencies of the auditory brainstem response (ABR). Design: The study tested ABRs in a rat animal model under isoflurane anesthesia. Study variables were duration of isoflurane anesthesia, stimulus frequency, stimulus level, and the dose of isoflurane. Rats were anesthetized with 1.5% or 2% isoflurane. ABRs were collected from 90 to 5 dB SPL at 5–40 kHz. Three full ABR series were collected over a 105-minute period. Thresholds were assigned, and ABR wave amplitudes and latencies were measured at each stimulus frequency and level. Study sample: Ten Sprague-Dawley rats were tested in a repeated measures design. Results: Statistical analyses revealed no significant effects of dose or duration on threshold, but a series of significant interactions between test variables for the amplitude and latency measurements. Conclusions: In the rat, dose and duration of isoflurane anesthesia induced inconsistent changes in latency and amplitude of the ABR. At 40 kHz, isoflurane dose had more powerful effects on latency and amplitude than occurred at other frequencies.
Key Words: Electrophysiology; isoflurane; auditory brainstem response; latency; rat
Isoflurane is a volatile gas anesthetic that is widely-used in auditory evoked potentials testing in humans and animals for research (Plourde & Picton, 1991; Richmond et al, 1996; Schwender et al, 1997; Johnson and Taylor, 1998; Rundshagen et al, 2002) and clinical purposes, including intra-operative monitoring during auditory nerve surgeries (Hsu et al, 1992; Lin et al, 1997). Studies have revealed significant changes in auditory processing induced by isoflurane anesthesia. Isoflurane can suppress the stapedius reflex (Makhdoum et al, 1998; Bissinger et al, 2000). Transient-evoked otoacoustic emissions (TEOAEs) were reduced in amplitude in human subjects, in comparison with unanesthetized TEOAE recordings. The effect occurred independently of blood pressure changes, suggesting a direct effect of isoflurane on cochlear processing (Ferber-Viart et al, 1998). No changes post-anesthesia have been detected in human TEOAEs (Buyukkocak et al, 2009), indicating that reductions in TEOAEs occur only during the active anesthetic phase with isoflurane, and do not represent permanent changes to the functioning of the peripheral auditory system. In C57Bl/6J mice, isoflurane (4% for induction, 1–1.5% for maintenance) induced 5–10 dB elevations of distortion product (DP)OAE thresholds over a 60-minute anesthetic period, compared to the initial baseline recordings taken immediately after anesthesia induction (Cederholm et al, 2012). Compared with the human and mouse results, discrepant results were detected from mustachioed bats under 1.5–2% isoflurane anesthesia. In the
bats, amplitudes of distortion product and spontaneous OAEs were increased under isoflurane anesthesia (4% induction, 1.5–2% maintenance) (Drexl et al, 2004). These data suggest the possibility that the impact of isoflurane on cochlear processing could be species- or class-specific. Isoflurane also has significant effects on auditory electrophysiology. In adult human surgical patients, isoflurane caused prolongations of the latencies of waves III and V of the auditory brainstem response (ABR), resulting in increased I-III, I-V, and III-V interpeak latencies (Manninen et al, 1985). Testing was performed at isoflurane levels of 1, 1.5, and 2%, along with a set of recordings in which 50% nitrous oxide was added to the isoflurane. The nitrous oxide had no effects on ABR latencies, nor did the dose of isoflurane. At all three dose levels (1, 1.5, 2%), the latencies were prolonged equally above the awake condition. Human auditory middle-latency responses can be significantly affected by isoflurane anesthesia (Schwender et al, 1994) and can be used as an index of depth of anesthesia (Heneghan et al, 1987). In Sprague-Dawley rats, isoflurane induced dose-dependent (0.38, 0.76, 1.13%) prolongations in all waves of the ABR, in comparison with awake recordings (Santarelli et al, 2003a,b). Stronks et al (2010) tested electrocochleography and ABRs from guinea pigs that were awake or anesthetized with 1–3% isoflurane. Compared with baselines from awake animals, 2% isoflurane induced amplitude reductions in the N2
Correspondence: Eric C. Bielefeld, Department of Speech and Hearing Science, The Ohio State University, 110 Pressey Hall, 1070 Carmack Road, Columbus, OH 43220, USA. E-mail: [email protected] (Received 26 June 2013; accepted 19 October 2013) ISSN 1499-2027 print/ISSN 1708-8186 online © 2014 British Society of Audiology, International Society of Audiology, and Nordic Audiological Society DOI: 10.3109/14992027.2013.858280
Bielefeld–Isoflurane and the ABR
Abbreviations
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ABR ANOVA CAP DPOAE TDT TEOAE
Auditory brainstem response Analysis of variance Compound action potential Distortion product otoacoustic emission Tucker Davis Technologies Transient-evoked otoacoustic emission
wave of the compound action potential (CAP), and 3% isoflurane reduced amplitudes of the N1 and N2 components of the CAP, as well as the cochlear microphonic. The 3% isoflurane also prolonged latencies and raised thresholds of the N1 and N2 components of the CAP. The later waves of the ABR, those with latencies longer than 4 ms, showed latency prolongations and amplitude reductions in a dose-dependent manner by the 2% and 3% isoflurane doses (Stronks et al, 2010). Auditory-evoked potentials thresholds are higher under isoflurane anesthesia than thresholds obtained under ketamine anesthesia in Long-Evans rats (Ruebausen et al, 2012) and the C129/Sv/Ev and C57Bl/6J mouse strains (Cederholm et al, 2012). In the C57Bl/6J mouse, the investigators also found a significant effect of the duration of isoflurane anesthesia on ABR threshold for click and 16-kHz tone burst stimuli. Thresholds were significantly higher for those stimuli after 60 minutes of isoflurane anesthesia compared to measures taken immediately after induction of anesthesia. This effect was not detected with ketamine anesthesia. The changes in sensitivity over the 60-minute interval of isoflurane anesthesia, as well as the reduced sensitivity compared to the recordings under ketamine anesthesia, were partly attributable to reductions in the amplitude input-output functions of the first two P-N complexes in the ABR waveforms. Because many clinical and research ABR protocols under isoflurane anesthesia are longer than 60 minutes in duration, the findings in the C57Bl/6J mouse that ABR thresholds and amplitudes change over time under isoflurane calls into question the validity of using isoflurane anesthesia when testing auditory evoked potentials. The current study was undertaken to further explore the duration effects of isoflurane on thresholds and amplitudes/latencies of the P1 and P2 wave in the Sprague-Dawley rat by assessing a broad range of audiometric frequencies, repeating ABR recordings over a longer interval, and repeating the test protocol under two different doses of isoflurane anesthesia. In doing so, the goal was to determine if ABR recordings change with the duration of isoflurane anesthesia. If the ABR does change over time, measurements taken early after induction of isoflurane anesthesia cannot be validly compared to measurements taken later. Conversely, if the ABRs do not change over time under anesthesia, it would provide evidence that measurements taken at any time point in the maintenance phase of isoflurane anesthesia can be compared to one another with appropriate validity.
Materials and Methods Subjects Ten adult male Sprague-Dawley rats were used in the study. They were obtained from Harlan Laboratories at ages 2–3 months. The animals were housed in a quiet colony (⬍ 45 dBA). All procedures involving use and care of the animals were reviewed and approved
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by The Ohio State University’s Institutional Animal Care and Use Committee.
Induction and maintenance of isoflurane anesthesia All animals were tested under isoflurane anesthesia. The isoflurane was delivered through a SurgiVet® tabletop research anesthesia machine with a Classic T3™ isoflurane funnel vaporizer. The oxygen delivery was controlled by the oxygen flowmeter on the anesthesia machine, which carries a range of 200 mL to 4 L/min oxygen flow rates. The oxygen flowmeter and isoflurane output levels are measured annually by the Ohio State University Laboratory Animal Resources, and calibrated by Smiths Medical (Dublin, Ohio, USA) if measurements reveal any problems. For the current study, the animals were initially placed in an acrylic induction box for three minutes with 4% isoflurane in O2 delivered at a 1 L/min flow rate. After the three-minute induction interval, the animals were moved to the testing booth, where they were placed on a homeothermic blanket (Harvard Apparatus, Hollisten, USA). A rectal thermometer connected directly to the homeothermic blanket monitored body temperature, and the controls for the blanket raised/lowered the heat on the blanket to keep the rat’s body temperature at 37 degrees Celsius. While in the testing booth, the rats received the maintenance level of isoflurane through a nose cone at 1.5% for the first series of trials. On a separate testing session held a minimum of three weeks after the first session, anesthesia was again induced with 4%, but then the maintenance level was 2%. Flow rate was held constant at 1 L/min throughout the duration of all tests.
ABR testing Each rat was tested on two separate occasions using free-field ABR. Needle recording electrodes were placed at the vertex (noninverting), below the left pinna (inverting) and behind the shoulder blade (ground). All stimuli were generated using Tucker Davis Technologies (TDT, Gainesville, USA) SigGen software. Each tone burst was 1 ms in duration, and had a 0.5 ms rise/fall time with no plateau. Stimuli were presented at a rate of 19/sec. Signals were routed to a speaker (TDT Model MF1) positioned at zero degrees azimuth, 10 cm from the vertex of each rat’s head. Acoustic stimuli were calibrated prior to each testing session, by recording the output of the speaker with a microphone (ACO Pacific 7016 ¼-inch microphone, ACO Pacific, Belmont, USA) placed at the animals’ head level, routing that through a pre-amplifier (ACO Pacific ACOustical Interface) to a measuring oscilloscope (Tektronix TDS 1012B, Tektonix, Inc., Beaverton, USA), and comparing the stimulus voltage to a 94 dB SPL stimulus from an acoustic calibrator (Larson Davis CAL200, Larson Davis, Depew, USA). The rats’ evoked responses were amplified with a gain of 50 000, using a TDT RA4LI headstage connected to an RA4PA pre-amplifier, and bandpass filtered from 0.1–3 kHz. Four hundred sweeps were averaged at each stimulus level using TDT BioSigRz software. The level of the signal was decreased in 5-dB steps from 90 dB SPL to 5 dB SPL in order to acquire ABR amplitude and latency input-output functions. Six frequencies were tested: 5, 10, 15, 20, 30, and 40 kHz. The duration of the testing for each series of all six frequencies (from 5 to 40 kHz) was 35 minutes. The testing of the series of six frequencies was repeated twice, for a total of three runs of the six test frequencies from 5 to 40 kHz (test times 1, 2, and 3). The order of frequencies was not randomized, so each set of recordings started with 5 kHz, and ended with 40 kHz. The total duration of the testing for the three 35-minute series was
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105 minutes, following the three minutes of anesthesia induction and the approximately two minutes of setting up the electrodes in the test booth. As described above, the first series of ABRs was collected while the rats were maintained under 1.5% isoflurane anesthesia. The second series, with the maintenance dose at 2%, was run on a separate testing session that was at least three weeks after the initial testing session. The minimum three-week interval was chosen to assure that the earlier dosing with 1.5% did not have any effect on the 2% testing session, and that the two testing sessions would be separate events that did not interact.
variance (ANOVA) was performed, with all three variables treated as repeated measures. In order to assess the effect of duration of isoflurane anesthesia on the latency and amplitude of the ABR, a series of three-way (time ⫻ stimulus level ⫻ dose) ANOVAs were performed for P1 and P2 latencies and amplitudes. Each frequency was analysed separately. All statistical analyses were performed using IBM SPSS Statistics v19.0.0. A p value of ⬍ 0.05 was considered significant. In cases of multiple comparisons using t-tests, a Bonferroni correction was applied to reduce Type I error.
Results
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ABR analyses In order to calibrate the responses acquired in the current experiment, data on amplitude voltages, thresholds, and latencies were compared with laboratory normative data for Sprague-Dawley and Fischer 344/ NHsd rats aged 2–4 months that were collected under isoflurane anesthesia. Since those normative data were collected under timing conditions equivalent to test time 1 of the current experiment, data collected in test time 1 (the first 35 minutes of testing) were those compared to the normative data. All test time 1 data collected in the current experiment were consistent with the previous normative data. Once each series of data was collected, the data file was coded by a graduate student so that the analyses of thresholds and amplitude/ latency input-output functions were done with the investigator blind to the test time and isoflurane dose condition during the analyses. For each ABR waveform series, a threshold was assigned. Threshold was recorded as the lowest level at which a detectable response of any ABR component was elicited and could be repeated. Following threshold assignment, each series was analysed for P1 and P2 amplitudes and latencies at each 5-dB step from 90 dB SPL down to threshold. The waveforms were analysed by placing cursors on the positive P1 and P2 peaks, along with the negative peaks that followed them. P1 was defined as the first positive peak in the waveform series, and occurred between 1.0 and 1.7 ms at 90 dB SPL. P2 was defined as the second positive peak, occurring between 0.5 and 1.0 ms after the P1 wave at 1.6–2.8 ms absolute latency for the 90 dB SPL stimulus.
Statistical analysis In order to assess the effect of isoflurane anesthesia on the thresholds of the ABR, a three-way (frequency ⫻ time ⫻ dose) analysis of
Effects of isoflurane anesthesia on threshold ABR threshold audiograms are displayed in Figure 1. Each audiogram shows three sets of recordings, reflecting times 1, 2, and 3. Panel A displays the 1.5% dose, and Panel B displays the separate testing session for the 2% dose. The three-factor repeated measure ANOVA (frequency ⫻ time ⫻ dose) revealed a significant two-way interaction of time ⫻ dose (p ⬍ 0.01). Subsequent analyses collapsed across frequency revealed no significant main effects of dose at any of the three test times, nor a main effect of test time at either of the two doses.
Effects of isoflurane anesthesia on P1 latency P1 latency input-output functions for the six frequencies examined in the study for the 1.5% dose condition and the 2% dose condition are displayed in Figure 2. The three-factor ANOVAs (time ⫻ stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant dose ⫻ time two-way interaction, indicating that the 1.5% and 2% doses differed from each other at least at one time point. Post hoc analyses collapsing across stimulus level revealed that the 2% dose had significantly longer latencies than the 1.5% dose at test time 3. At 20 kHz, there was a significant three-way interaction. A series of two-factor (time ⫻ dose) ANOVAs were run at each stimulus level. Those tests revealed a significant interaction at 90 dB SPL, indicating that the 1.5% and 2% doses differed from each other at least at one time point, only at that 90 dB SPL stimulus level. A series of 6 paired samples t-tests with Bonferroni correction (corrected significant p value ⬍ 0.0083) was run to compare doses in each test time interval at 90 dB SPL. They revealed that the 2% dose had longer
Figure 1. Mean ABR thresholds for test times 1–3 at the six frequencies tested. (A) 1.5% isoflurane dose condition. (B) 2% isoflurane dose condition. Error bars are ⫾ 1 SEM.
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Bielefeld–Isoflurane and the ABR
Figure 2. Mean P1 latencies for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean latencies are plotted linearly on the y-axis. Error bars are ⫾ 1 SEM. Note that the y-axis scale for the 40 kHz plot is broader than the others due to the longer latencies of those P1 waves.
latencies at 90 dB SPL in the test time 3 compared with test time 3 of the 1.5% condition. At 30 kHz, there was a significant dose ⫻ stimulus level two-way interaction, indicating that the 1.5% and 2% doses were different from each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni correction (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The 1.5% dose had significantly longer latencies than the 2% dose at the 35 dB SPL stimulus level. There were no significant differences at any other stimulus level. At 40 kHz, there was a significant test time ⫻ stimulus level interaction, indicating that at least two test times were different from each other at a minimum
of one stimulus level, regardless of the dose of isoflurane. One-way ANOVAs with t-test post hoc analyses were run at each stimulus level, and revealed that test time 1 in both the 1.5% and 2% dose conditions showed longer latencies than test times 2 or 3 at the 70– 75 dB SPL stimulus levels.
Effects of isoflurane anesthesia on P1 amplitude P1 amplitude input-output functions for the six frequencies examined in the study for the 1.5% dose condition and the 2% dose condition are displayed in Figure 3. As with the P1 latency measurements, the three-factor ANOVAs (test time ⫻ stimulus level ⫻ dose) at
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Figure 3. Mean P1 amplitudes for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean latencies are plotted linearly on the y-axis. Error bars are ⫾ 1 SEM.
each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant three-way interaction. Two-factor ANOVAs (test time ⫻ dose) at each stimulus level revealed significant main effects of test time at the 75, 80, and 90 dB SPL stimulus levels. The main effects resulted from test time 1 showing higher amplitudes than times 2 or 3, regardless of dose. At the 70 and 85 dB SPL stimulus levels, there were two-way test time ⫻ dose interactions, indicating that at least two of the test times differed from each other at 1.5% or 2%. Analysis of those interactions revealed that test time 1 had higher amplitudes than times 2 and 3, only in the 2% dose condition at both 70 and 85 dB SPL. At 10 kHz, there
was a trend (p ⫽ 0.053) toward a two-way interaction of dose and stimulus level. At 15 kHz, there was a significant dose ⫻ stimulus level two-way interaction, indicating that the 1.5% and 2% doses were different from each other at some stimulus levels, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The doses were different at only the 70 dB SPL stimulus level, with the 1.5% dose showing lower amplitudes at that level. At 40 kHz, there was a significant dose ⫻ stimulus level interaction, again indicating that the 1.5% and 2% doses were different from
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Bielefeld–Isoflurane and the ABR
Figure 4. Mean P2 latencies for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean amplitudes are plotted logarithmically on the y-axis. Error bars are ⫾ 1 SEM. Note that the y-axis scale for the 40 kHz plot is broader than the others due to the longer latencies of those P2 waves.
each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The 2% dose showed higher amplitudes than 1.5% at 60, 65, 70, and 75 dB SPL.
Effects of isoflurane anesthesia on P2 latency P2 latency input-output functions are displayed in Figure 4. As with the previous measurements, the three-factor ANOVAs (test time ⫻
stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 10 kHz, there was a significant two-way interaction of test time ⫻ dose, indicating that at least two of the test times differed from each other at 1.5% or 2%, regardless of stimulus level. A two-factor ANOVA (test time ⫻ dose) collapsing across stimulus level revealed a significant two-way interaction. One-factor ANOVAs with post hoc t-tests comparing test times at each dose revealed that test time 3 had shorter latencies than times 1 or 2 in the 1.5% dose condition only. At 20 kHz, there was a significant dose ⫻ stimulus level two-way interaction, again
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Figure 5. Mean P2 amplitudes for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean amplitudes are plotted logarithmically on the y-axis. Error bars are ⫾ 1 SEM.
indicating that the 1.5% and 2% doses were different from each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The doses were different at only the 60 dB SPL stimulus level, with the 1.5% condition showing longer latencies than the 2% condition. At 40 kHz, there was also a significant dose ⫻ stimulus level twoway interaction. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level, and revealed that the 1.5% dose had longer latencies than the 2% condition at the 60, 65, and 70 dB SPL stimulus levels.
Effects of isoflurane anesthesia on P2 amplitudes P2 amplitude input-output functions are displayed in Figure 5. As with the previous measurements, the three-factor ANOVAs (test time ⫻ stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant three-way interaction. A series of two-factor ANOVAs (test time ⫻ dose) was run at each stimulus level. The ANOVAs revealed a variety of significant main effects. At 30–50 dB SPL, there were significant main effects of dose. Collapsing across test times, t-tests at each stimulus level revealed that the 2% dose condition has higher amplitudes than the 1.5% condition at 30–50 dB SPL. At the 60– 80 dB SPL stimulus levels, the two-factor ANOVAs revealed significant main effects of test time. Collapsing across dose, one-factor
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Bielefeld–Isoflurane and the ABR ANOVAs revealed that test time 1 had higher amplitudes than times 2 or 3 at the 60–80 dB SPL stimulus levels. At 10 kHz, there were trends (p ⫽ 0.057 and p ⫽ 0.059) toward a two-way interaction of dose ⫻ stimulus level and a three-way interaction. At 15 kHz, there was a significant three-way interaction. A series of two-factor ANOVAs (test time ⫻ dose) was run at each stimulus level. At the 70 and 75 dB SPL stimulus levels, there was a significant main effect of dose. Post hoc t-testing revealed that the 2% dose had higher amplitudes than the 1.5% condition at the 70 and 75 dB SPL levels. The two-factor ANOVA also revealed significant main effects of test time at the 85 and 90 dB SPL stimulus. Post hoc analyses revealed that test time 3 had higher amplitudes than time 2 at 85 and 90 dB SPL. At 30 kHz, there was a three-way interaction. A series of twofactor ANOVAs (test time ⫻ dose) was run at each stimulus level. The only notable outcome was a statistical trend (p ⫽ 0.068) toward a two-way interaction of dose and test time at the 90 dB SPL stimulus level. At 40 kHz, there was only a trend (p ⫽ 0.051) toward a dose ⫻ stimulus level interaction.
Discussion The current study was undertaken to address the issue of whether the duration of isoflurane anesthesia will affect internal, repeated measures studies of the auditory system. If there was a significant impact of isoflurane anesthesia duration, it would render invalid comparisons between any data that were collected at different time points after anesthesia induction. With the significant impact that isoflurane anesthesia can have on OAEs (Ferber-Viart et al, 1998; Cederholm et al, 2012) and auditory evoked potential thresholds (Ruebhausen et al, 2012), the possibility existed that prolonged anesthetic state under isoflurane would lead to significant variations in ABR thresholds, amplitudes, and latencies. Dose of isoflurane has also been shown to have effects on auditory-evoked potential waveforms (Santarelli et al, 2003a,b; Stronks et al, 2010). Therefore, two doses, 1.5% and 2% isoflurane, were examined in the current study to see if the ABRs collected under the two doses differed from each other, and if effects of duration of isoflurane anesthesia were dependent upon dose level. As detailed in the Results, there were no significant effects of duration on thresholds, but there were a number of statistically-significant effects of anesthesia duration (as indexed by the two-way and three-way interactions detailed in the Results) on latency and amplitude of the P1 and P2 waves. Although the differences were often statistically significant, post hoc analyses of the interactions did not show a stable or consistent pattern across the different frequencies tested or test metrics used. The data reported by Cederholm et al (2012) that were obtained from the C57Bl/6J mouse show a consistent effect of reduction in ABR component amplitudes over a 60-minute period of isoflurane anesthesia. Such a consistent effect was not found in the current investigation. The analysis of the large number of variables and testing conditions in the current study was likely to yield some random significant results, and that appears to be the case with the significant effects that were detailed in the Results section. Many of the post hoc analyses of the significant interactions found in the current study resulted in single test times being different from each other at only single stimulus levels, suggesting that the differences were random fluctuations rather than truly impactful findings that would require investigators to review past data collected under isoflurane anesthesia or alter future testing protocols. The current experiment utilized spontaneous breathing during the induction and maintenance of isoflurane anesthesia, as opposed
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to mechanical ventilation as was used in human surgical protocols (Manninen et al, 1985; Ferber-Viart et al, 1998; Buyukkocak et al, 2009) and was used in the Stronks et al (2010) study of the guinea pig. Mechanical ventilation provides a more stable anesthetic level in the respiratory system than spontaneous inhalation does. In conditions of spontaneous breathing, such as in the current study, the induction of anesthetic state can lead to changes in breathing rates. A reduction in breathing rate would subsequently lead to a decrease in isoflurane in the animal’s system. The uncertainty in exposure levels to the isoflurane when delivered through spontaneous breathing could account for some of the variability seen between animals in the current study, as well as the inconsistent effects of the two different doses on the waveform latencies and amplitudes. Whether the difference in outcomes between the current study and the work by Cederholm et al (2012), which also utilized spontaneous breathing, could be due to difference in species or due to the specific differences in protocols used is unknown. Two doses of isoflurane were utilized in the current study, 1.5 and 2%. The expectation was that, if there was any effect of dose, the 2% dose would suppress amplitudes, and possibly prolong latencies, relative to the 1.5% dosing condition. Also, a duration effect seemed more likely to occur in the 2% condition than in the 1.5% condition, since the total amount of inhaled isoflurane would be considerably greater. As was the case with an effect of duration, no consistent significant differences appeared between the two dosing conditions. For example, the effects of dose that were seen in the amplitude measures sometimes saw the 2% condition showing lower amplitudes than the 1.5% condition, but then sometimes showing higher amplitudes than the 1.5% condition. Dose had very few effects on latency, other than reducing the variances between test times for the latencies of P1. The variances were generally lower in the 2% condition, suggesting more stable responses across test times and stimulus levels in comparison with the 1.5% condition. As can be seen in Figures 2–5, the variances are generally smaller for the 2% condition than the 1.5% condition. The most compelling evidence for a significant impact of isoflurane on the ABR was the testing performed at 40 kHz. In the Fischer 344/NHsd rat, 40 kHz is often more variable from animal to animal and test to test in terms of the threshold and ABR input-output functions (Bielefeld et al, 2008a,b), and the Sprague-Dawley appears to be no different. Thresholds were highest at 40 kHz, regardless of test time or dose condition (Figure 1). The error bars for the 40 kHz graphs in Figures 2–5 are consistently larger than other frequencies, indicating greater variability between animals and within the repeated measures for each of the tests. The 40 kHz frequency showed a significant interaction for the P1 and P2 latency measures, and for P1 amplitude. The P1 amplitude effect of isoflurane dose was also among largest differences that affected the highest number of stimulus levels (Figure 3). But like other frequencies and measurements, the effect of isoflurane dose on the P1 amplitude at 40 kHz was not consistent.
Conclusion Although numerous statistically significant effects were detected when comparing duration, dose, and stimulus level at each test frequency, there was no consistent effect on latency or amplitude of the ABR components. Combined with the lack of significant effect on thresholds, the current data indicate that duration of isoflurane anesthesia is not a significant factor for ABR testing in the SpragueDawley rat, at least up to the ~110 minutes of anesthetic state tested
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in the current study. ABR measurements collected within that time frame appear to be equally valid and suitable for comparisons to one another. A longer duration may indeed begin to impact the ABR, as could higher dose levels of isoflurane than those tested in the current study.
Acknowledgements
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The author thanks Ellen Hambley, José Quilles, Katherine Kerns, and Meghan Joyce for their assistance with the data collection and analyses. Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
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Hsu J.C., Lui T.N., Yu C.L., Chen Y.C., Chang C.N. et al. 1992. The simultaneous use of electrocochleogram, brainstem auditory-evoked potential and facial muscle EMG in cerebellopontine angle tumor removal. J Formos Med Assoc, 91, 580–584. Johnson C.B. & Taylor P.M. 1998. Comparison of the effects of halothane, isoflurane and methoxyflurane on the electroencephalogram of the horse. Br J Anaesth, 81, 748–753. Lin C.M., Hsu J.C., Wu R.S., Wu K.C., Yu C.L. et al. 1997. Evoked facial nerve EMG and brainstem auditory-evoked potential monitoring in cerebellopontine angle tumor resection. Acta Anaesthesiol Sin, 35, 141–147. Makhdoum M.J., Snik A.F., Stollman M.H., de Grood P.M. & van den Broek P. 1998. The influence of the concentration of volatile anesthetics on the stapedius reflex determined intraoperatively during cochlear implantation in children. Am J Otol, 19, 598–603. Manninen P.H., Lam A.M. & Nicholas J.F. 1985. The effects of isoflurane and isoflurane - nitrous oxide anesthesia on brainstem auditory evoked potentials in humans. Anesth Analg, 64, 43–47. Plourde G. & Picton T.W. 1991. Long-latency auditory-evoked potentials during general anesthesia: N1 and P3 components. Anesth Analg, 72, 342–350. Richmond C.E., Matson A., Thornton C., Doré C.J. & Newton D.E. 1996. Effect of neuromuscular block on depth of anaesthesia as measured by the auditory-evoked response. Br J Anaesth, 76, 446–448. Ruebhausen M.R., Brozoski T.J. & Bauer C.A. 2012. A comparison of the effects of isoflurane and ketamine anesthesia on auditory brainstem response (ABR) thresholds in rats. Hear Res, 287, 25–29. Rundshagen I., Schnabel K. & Schulte am Esch J. 2002. Recovery of memory after general anaesthesia: Clinical findings and somatosensory-evoked responses. Br J Anaesth, 88, 362–368. Santarelli R., Carraro L., Conti G., Capello M., Plourde G. et al. 2003. Effects of isoflurane on auditory middle latency (MLRs) and steady-state (SSRs) responses recorded from the temporal cortex of the rat. Brain Res, 973, 240–251. Santarelli R., Arslan E., Carraro L., Conti G., Capello M. et al. 2003. Effects of isoflurane on the auditory brainstem responses and middle latency responses of rats. Acta Otolaryngol, 123, 176–181. Schwender D., Daunderer M., Mulzer S., Klasing S., Finsterer U. et al. 1997. Mid-latency auditory-evoked potentials predict movements during anesthesia with isoflurane or propofol. Anesth Analg, 85, 164–173. Schwender D., Kaiser A., Klasing S., Peter K. & Pöppel E. 1994. Mid-latency auditory-evoked potentials and explicit and implicit memory in patients undergoing cardiac surgery. Anesthesiology, 80, 493–501. Stronks H.C., Aarts M.C.J. & Klis S.F.L. 2010. Effects of isoflurane on auditory-evoked potentials in the cochlea and brainstem of guinea pigs. Hear Res, 260, 20–29.
Original Article
Influence of dose and duration of isoflurane anesthesia on the auditory brainstem response in the rat
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Eric C. Bielefeld Department of Speech and Hearing Science, The Ohio State University, Columbus, Ohio, USA
Abstract Objective: Isoflurane anesthesia can have significant effects on processing of sounds at the peripheral and central levels, manifesting in changes in auditory-evoked potentials. The current study tested whether duration of isoflurane anesthesia changes thresholds, amplitudes, and latencies of the auditory brainstem response (ABR). Design: The study tested ABRs in a rat animal model under isoflurane anesthesia. Study variables were duration of isoflurane anesthesia, stimulus frequency, stimulus level, and the dose of isoflurane. Rats were anesthetized with 1.5% or 2% isoflurane. ABRs were collected from 90 to 5 dB SPL at 5–40 kHz. Three full ABR series were collected over a 105-minute period. Thresholds were assigned, and ABR wave amplitudes and latencies were measured at each stimulus frequency and level. Study sample: Ten Sprague-Dawley rats were tested in a repeated measures design. Results: Statistical analyses revealed no significant effects of dose or duration on threshold, but a series of significant interactions between test variables for the amplitude and latency measurements. Conclusions: In the rat, dose and duration of isoflurane anesthesia induced inconsistent changes in latency and amplitude of the ABR. At 40 kHz, isoflurane dose had more powerful effects on latency and amplitude than occurred at other frequencies.
Key Words: Electrophysiology; isoflurane; auditory brainstem response; latency; rat
Isoflurane is a volatile gas anesthetic that is widely-used in auditory evoked potentials testing in humans and animals for research (Plourde & Picton, 1991; Richmond et al, 1996; Schwender et al, 1997; Johnson and Taylor, 1998; Rundshagen et al, 2002) and clinical purposes, including intra-operative monitoring during auditory nerve surgeries (Hsu et al, 1992; Lin et al, 1997). Studies have revealed significant changes in auditory processing induced by isoflurane anesthesia. Isoflurane can suppress the stapedius reflex (Makhdoum et al, 1998; Bissinger et al, 2000). Transient-evoked otoacoustic emissions (TEOAEs) were reduced in amplitude in human subjects, in comparison with unanesthetized TEOAE recordings. The effect occurred independently of blood pressure changes, suggesting a direct effect of isoflurane on cochlear processing (Ferber-Viart et al, 1998). No changes post-anesthesia have been detected in human TEOAEs (Buyukkocak et al, 2009), indicating that reductions in TEOAEs occur only during the active anesthetic phase with isoflurane, and do not represent permanent changes to the functioning of the peripheral auditory system. In C57Bl/6J mice, isoflurane (4% for induction, 1–1.5% for maintenance) induced 5–10 dB elevations of distortion product (DP)OAE thresholds over a 60-minute anesthetic period, compared to the initial baseline recordings taken immediately after anesthesia induction (Cederholm et al, 2012). Compared with the human and mouse results, discrepant results were detected from mustachioed bats under 1.5–2% isoflurane anesthesia. In the
bats, amplitudes of distortion product and spontaneous OAEs were increased under isoflurane anesthesia (4% induction, 1.5–2% maintenance) (Drexl et al, 2004). These data suggest the possibility that the impact of isoflurane on cochlear processing could be species- or class-specific. Isoflurane also has significant effects on auditory electrophysiology. In adult human surgical patients, isoflurane caused prolongations of the latencies of waves III and V of the auditory brainstem response (ABR), resulting in increased I-III, I-V, and III-V interpeak latencies (Manninen et al, 1985). Testing was performed at isoflurane levels of 1, 1.5, and 2%, along with a set of recordings in which 50% nitrous oxide was added to the isoflurane. The nitrous oxide had no effects on ABR latencies, nor did the dose of isoflurane. At all three dose levels (1, 1.5, 2%), the latencies were prolonged equally above the awake condition. Human auditory middle-latency responses can be significantly affected by isoflurane anesthesia (Schwender et al, 1994) and can be used as an index of depth of anesthesia (Heneghan et al, 1987). In Sprague-Dawley rats, isoflurane induced dose-dependent (0.38, 0.76, 1.13%) prolongations in all waves of the ABR, in comparison with awake recordings (Santarelli et al, 2003a,b). Stronks et al (2010) tested electrocochleography and ABRs from guinea pigs that were awake or anesthetized with 1–3% isoflurane. Compared with baselines from awake animals, 2% isoflurane induced amplitude reductions in the N2
Correspondence: Eric C. Bielefeld, Department of Speech and Hearing Science, The Ohio State University, 110 Pressey Hall, 1070 Carmack Road, Columbus, OH 43220, USA. E-mail: [email protected] (Received 26 June 2013; accepted 19 October 2013) ISSN 1499-2027 print/ISSN 1708-8186 online © 2014 British Society of Audiology, International Society of Audiology, and Nordic Audiological Society DOI: 10.3109/14992027.2013.858280
Bielefeld–Isoflurane and the ABR
Abbreviations
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ABR ANOVA CAP DPOAE TDT TEOAE
Auditory brainstem response Analysis of variance Compound action potential Distortion product otoacoustic emission Tucker Davis Technologies Transient-evoked otoacoustic emission
wave of the compound action potential (CAP), and 3% isoflurane reduced amplitudes of the N1 and N2 components of the CAP, as well as the cochlear microphonic. The 3% isoflurane also prolonged latencies and raised thresholds of the N1 and N2 components of the CAP. The later waves of the ABR, those with latencies longer than 4 ms, showed latency prolongations and amplitude reductions in a dose-dependent manner by the 2% and 3% isoflurane doses (Stronks et al, 2010). Auditory-evoked potentials thresholds are higher under isoflurane anesthesia than thresholds obtained under ketamine anesthesia in Long-Evans rats (Ruebausen et al, 2012) and the C129/Sv/Ev and C57Bl/6J mouse strains (Cederholm et al, 2012). In the C57Bl/6J mouse, the investigators also found a significant effect of the duration of isoflurane anesthesia on ABR threshold for click and 16-kHz tone burst stimuli. Thresholds were significantly higher for those stimuli after 60 minutes of isoflurane anesthesia compared to measures taken immediately after induction of anesthesia. This effect was not detected with ketamine anesthesia. The changes in sensitivity over the 60-minute interval of isoflurane anesthesia, as well as the reduced sensitivity compared to the recordings under ketamine anesthesia, were partly attributable to reductions in the amplitude input-output functions of the first two P-N complexes in the ABR waveforms. Because many clinical and research ABR protocols under isoflurane anesthesia are longer than 60 minutes in duration, the findings in the C57Bl/6J mouse that ABR thresholds and amplitudes change over time under isoflurane calls into question the validity of using isoflurane anesthesia when testing auditory evoked potentials. The current study was undertaken to further explore the duration effects of isoflurane on thresholds and amplitudes/latencies of the P1 and P2 wave in the Sprague-Dawley rat by assessing a broad range of audiometric frequencies, repeating ABR recordings over a longer interval, and repeating the test protocol under two different doses of isoflurane anesthesia. In doing so, the goal was to determine if ABR recordings change with the duration of isoflurane anesthesia. If the ABR does change over time, measurements taken early after induction of isoflurane anesthesia cannot be validly compared to measurements taken later. Conversely, if the ABRs do not change over time under anesthesia, it would provide evidence that measurements taken at any time point in the maintenance phase of isoflurane anesthesia can be compared to one another with appropriate validity.
Materials and Methods Subjects Ten adult male Sprague-Dawley rats were used in the study. They were obtained from Harlan Laboratories at ages 2–3 months. The animals were housed in a quiet colony (⬍ 45 dBA). All procedures involving use and care of the animals were reviewed and approved
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by The Ohio State University’s Institutional Animal Care and Use Committee.
Induction and maintenance of isoflurane anesthesia All animals were tested under isoflurane anesthesia. The isoflurane was delivered through a SurgiVet® tabletop research anesthesia machine with a Classic T3™ isoflurane funnel vaporizer. The oxygen delivery was controlled by the oxygen flowmeter on the anesthesia machine, which carries a range of 200 mL to 4 L/min oxygen flow rates. The oxygen flowmeter and isoflurane output levels are measured annually by the Ohio State University Laboratory Animal Resources, and calibrated by Smiths Medical (Dublin, Ohio, USA) if measurements reveal any problems. For the current study, the animals were initially placed in an acrylic induction box for three minutes with 4% isoflurane in O2 delivered at a 1 L/min flow rate. After the three-minute induction interval, the animals were moved to the testing booth, where they were placed on a homeothermic blanket (Harvard Apparatus, Hollisten, USA). A rectal thermometer connected directly to the homeothermic blanket monitored body temperature, and the controls for the blanket raised/lowered the heat on the blanket to keep the rat’s body temperature at 37 degrees Celsius. While in the testing booth, the rats received the maintenance level of isoflurane through a nose cone at 1.5% for the first series of trials. On a separate testing session held a minimum of three weeks after the first session, anesthesia was again induced with 4%, but then the maintenance level was 2%. Flow rate was held constant at 1 L/min throughout the duration of all tests.
ABR testing Each rat was tested on two separate occasions using free-field ABR. Needle recording electrodes were placed at the vertex (noninverting), below the left pinna (inverting) and behind the shoulder blade (ground). All stimuli were generated using Tucker Davis Technologies (TDT, Gainesville, USA) SigGen software. Each tone burst was 1 ms in duration, and had a 0.5 ms rise/fall time with no plateau. Stimuli were presented at a rate of 19/sec. Signals were routed to a speaker (TDT Model MF1) positioned at zero degrees azimuth, 10 cm from the vertex of each rat’s head. Acoustic stimuli were calibrated prior to each testing session, by recording the output of the speaker with a microphone (ACO Pacific 7016 ¼-inch microphone, ACO Pacific, Belmont, USA) placed at the animals’ head level, routing that through a pre-amplifier (ACO Pacific ACOustical Interface) to a measuring oscilloscope (Tektronix TDS 1012B, Tektonix, Inc., Beaverton, USA), and comparing the stimulus voltage to a 94 dB SPL stimulus from an acoustic calibrator (Larson Davis CAL200, Larson Davis, Depew, USA). The rats’ evoked responses were amplified with a gain of 50 000, using a TDT RA4LI headstage connected to an RA4PA pre-amplifier, and bandpass filtered from 0.1–3 kHz. Four hundred sweeps were averaged at each stimulus level using TDT BioSigRz software. The level of the signal was decreased in 5-dB steps from 90 dB SPL to 5 dB SPL in order to acquire ABR amplitude and latency input-output functions. Six frequencies were tested: 5, 10, 15, 20, 30, and 40 kHz. The duration of the testing for each series of all six frequencies (from 5 to 40 kHz) was 35 minutes. The testing of the series of six frequencies was repeated twice, for a total of three runs of the six test frequencies from 5 to 40 kHz (test times 1, 2, and 3). The order of frequencies was not randomized, so each set of recordings started with 5 kHz, and ended with 40 kHz. The total duration of the testing for the three 35-minute series was
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105 minutes, following the three minutes of anesthesia induction and the approximately two minutes of setting up the electrodes in the test booth. As described above, the first series of ABRs was collected while the rats were maintained under 1.5% isoflurane anesthesia. The second series, with the maintenance dose at 2%, was run on a separate testing session that was at least three weeks after the initial testing session. The minimum three-week interval was chosen to assure that the earlier dosing with 1.5% did not have any effect on the 2% testing session, and that the two testing sessions would be separate events that did not interact.
variance (ANOVA) was performed, with all three variables treated as repeated measures. In order to assess the effect of duration of isoflurane anesthesia on the latency and amplitude of the ABR, a series of three-way (time ⫻ stimulus level ⫻ dose) ANOVAs were performed for P1 and P2 latencies and amplitudes. Each frequency was analysed separately. All statistical analyses were performed using IBM SPSS Statistics v19.0.0. A p value of ⬍ 0.05 was considered significant. In cases of multiple comparisons using t-tests, a Bonferroni correction was applied to reduce Type I error.
Results
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ABR analyses In order to calibrate the responses acquired in the current experiment, data on amplitude voltages, thresholds, and latencies were compared with laboratory normative data for Sprague-Dawley and Fischer 344/ NHsd rats aged 2–4 months that were collected under isoflurane anesthesia. Since those normative data were collected under timing conditions equivalent to test time 1 of the current experiment, data collected in test time 1 (the first 35 minutes of testing) were those compared to the normative data. All test time 1 data collected in the current experiment were consistent with the previous normative data. Once each series of data was collected, the data file was coded by a graduate student so that the analyses of thresholds and amplitude/ latency input-output functions were done with the investigator blind to the test time and isoflurane dose condition during the analyses. For each ABR waveform series, a threshold was assigned. Threshold was recorded as the lowest level at which a detectable response of any ABR component was elicited and could be repeated. Following threshold assignment, each series was analysed for P1 and P2 amplitudes and latencies at each 5-dB step from 90 dB SPL down to threshold. The waveforms were analysed by placing cursors on the positive P1 and P2 peaks, along with the negative peaks that followed them. P1 was defined as the first positive peak in the waveform series, and occurred between 1.0 and 1.7 ms at 90 dB SPL. P2 was defined as the second positive peak, occurring between 0.5 and 1.0 ms after the P1 wave at 1.6–2.8 ms absolute latency for the 90 dB SPL stimulus.
Statistical analysis In order to assess the effect of isoflurane anesthesia on the thresholds of the ABR, a three-way (frequency ⫻ time ⫻ dose) analysis of
Effects of isoflurane anesthesia on threshold ABR threshold audiograms are displayed in Figure 1. Each audiogram shows three sets of recordings, reflecting times 1, 2, and 3. Panel A displays the 1.5% dose, and Panel B displays the separate testing session for the 2% dose. The three-factor repeated measure ANOVA (frequency ⫻ time ⫻ dose) revealed a significant two-way interaction of time ⫻ dose (p ⬍ 0.01). Subsequent analyses collapsed across frequency revealed no significant main effects of dose at any of the three test times, nor a main effect of test time at either of the two doses.
Effects of isoflurane anesthesia on P1 latency P1 latency input-output functions for the six frequencies examined in the study for the 1.5% dose condition and the 2% dose condition are displayed in Figure 2. The three-factor ANOVAs (time ⫻ stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant dose ⫻ time two-way interaction, indicating that the 1.5% and 2% doses differed from each other at least at one time point. Post hoc analyses collapsing across stimulus level revealed that the 2% dose had significantly longer latencies than the 1.5% dose at test time 3. At 20 kHz, there was a significant three-way interaction. A series of two-factor (time ⫻ dose) ANOVAs were run at each stimulus level. Those tests revealed a significant interaction at 90 dB SPL, indicating that the 1.5% and 2% doses differed from each other at least at one time point, only at that 90 dB SPL stimulus level. A series of 6 paired samples t-tests with Bonferroni correction (corrected significant p value ⬍ 0.0083) was run to compare doses in each test time interval at 90 dB SPL. They revealed that the 2% dose had longer
Figure 1. Mean ABR thresholds for test times 1–3 at the six frequencies tested. (A) 1.5% isoflurane dose condition. (B) 2% isoflurane dose condition. Error bars are ⫾ 1 SEM.
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Bielefeld–Isoflurane and the ABR
Figure 2. Mean P1 latencies for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean latencies are plotted linearly on the y-axis. Error bars are ⫾ 1 SEM. Note that the y-axis scale for the 40 kHz plot is broader than the others due to the longer latencies of those P1 waves.
latencies at 90 dB SPL in the test time 3 compared with test time 3 of the 1.5% condition. At 30 kHz, there was a significant dose ⫻ stimulus level two-way interaction, indicating that the 1.5% and 2% doses were different from each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni correction (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The 1.5% dose had significantly longer latencies than the 2% dose at the 35 dB SPL stimulus level. There were no significant differences at any other stimulus level. At 40 kHz, there was a significant test time ⫻ stimulus level interaction, indicating that at least two test times were different from each other at a minimum
of one stimulus level, regardless of the dose of isoflurane. One-way ANOVAs with t-test post hoc analyses were run at each stimulus level, and revealed that test time 1 in both the 1.5% and 2% dose conditions showed longer latencies than test times 2 or 3 at the 70– 75 dB SPL stimulus levels.
Effects of isoflurane anesthesia on P1 amplitude P1 amplitude input-output functions for the six frequencies examined in the study for the 1.5% dose condition and the 2% dose condition are displayed in Figure 3. As with the P1 latency measurements, the three-factor ANOVAs (test time ⫻ stimulus level ⫻ dose) at
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Figure 3. Mean P1 amplitudes for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean latencies are plotted linearly on the y-axis. Error bars are ⫾ 1 SEM.
each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant three-way interaction. Two-factor ANOVAs (test time ⫻ dose) at each stimulus level revealed significant main effects of test time at the 75, 80, and 90 dB SPL stimulus levels. The main effects resulted from test time 1 showing higher amplitudes than times 2 or 3, regardless of dose. At the 70 and 85 dB SPL stimulus levels, there were two-way test time ⫻ dose interactions, indicating that at least two of the test times differed from each other at 1.5% or 2%. Analysis of those interactions revealed that test time 1 had higher amplitudes than times 2 and 3, only in the 2% dose condition at both 70 and 85 dB SPL. At 10 kHz, there
was a trend (p ⫽ 0.053) toward a two-way interaction of dose and stimulus level. At 15 kHz, there was a significant dose ⫻ stimulus level two-way interaction, indicating that the 1.5% and 2% doses were different from each other at some stimulus levels, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The doses were different at only the 70 dB SPL stimulus level, with the 1.5% dose showing lower amplitudes at that level. At 40 kHz, there was a significant dose ⫻ stimulus level interaction, again indicating that the 1.5% and 2% doses were different from
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Bielefeld–Isoflurane and the ABR
Figure 4. Mean P2 latencies for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean amplitudes are plotted logarithmically on the y-axis. Error bars are ⫾ 1 SEM. Note that the y-axis scale for the 40 kHz plot is broader than the others due to the longer latencies of those P2 waves.
each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The 2% dose showed higher amplitudes than 1.5% at 60, 65, 70, and 75 dB SPL.
Effects of isoflurane anesthesia on P2 latency P2 latency input-output functions are displayed in Figure 4. As with the previous measurements, the three-factor ANOVAs (test time ⫻
stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 10 kHz, there was a significant two-way interaction of test time ⫻ dose, indicating that at least two of the test times differed from each other at 1.5% or 2%, regardless of stimulus level. A two-factor ANOVA (test time ⫻ dose) collapsing across stimulus level revealed a significant two-way interaction. One-factor ANOVAs with post hoc t-tests comparing test times at each dose revealed that test time 3 had shorter latencies than times 1 or 2 in the 1.5% dose condition only. At 20 kHz, there was a significant dose ⫻ stimulus level two-way interaction, again
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Figure 5. Mean P2 amplitudes for test times 1–3 in the 1.5% and 2% dose conditions. Data are presented as a function of stimulus level for the 5, 10, 15, 20, 30, and 40 kHz test frequencies. Mean amplitudes are plotted logarithmically on the y-axis. Error bars are ⫾ 1 SEM.
indicating that the 1.5% and 2% doses were different from each other at least at one stimulus level, regardless of the test time. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level. The doses were different at only the 60 dB SPL stimulus level, with the 1.5% condition showing longer latencies than the 2% condition. At 40 kHz, there was also a significant dose ⫻ stimulus level twoway interaction. Collapsing across test times, a series of 13 paired samples t-tests with Bonferroni corrections (corrected significant p value ⬍ 0.0038) compared the two dose conditions at each stimulus level, and revealed that the 1.5% dose had longer latencies than the 2% condition at the 60, 65, and 70 dB SPL stimulus levels.
Effects of isoflurane anesthesia on P2 amplitudes P2 amplitude input-output functions are displayed in Figure 5. As with the previous measurements, the three-factor ANOVAs (test time ⫻ stimulus level ⫻ dose) at each frequency revealed several statistically significant interactions. At 5 kHz, there was a significant three-way interaction. A series of two-factor ANOVAs (test time ⫻ dose) was run at each stimulus level. The ANOVAs revealed a variety of significant main effects. At 30–50 dB SPL, there were significant main effects of dose. Collapsing across test times, t-tests at each stimulus level revealed that the 2% dose condition has higher amplitudes than the 1.5% condition at 30–50 dB SPL. At the 60– 80 dB SPL stimulus levels, the two-factor ANOVAs revealed significant main effects of test time. Collapsing across dose, one-factor
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Bielefeld–Isoflurane and the ABR ANOVAs revealed that test time 1 had higher amplitudes than times 2 or 3 at the 60–80 dB SPL stimulus levels. At 10 kHz, there were trends (p ⫽ 0.057 and p ⫽ 0.059) toward a two-way interaction of dose ⫻ stimulus level and a three-way interaction. At 15 kHz, there was a significant three-way interaction. A series of two-factor ANOVAs (test time ⫻ dose) was run at each stimulus level. At the 70 and 75 dB SPL stimulus levels, there was a significant main effect of dose. Post hoc t-testing revealed that the 2% dose had higher amplitudes than the 1.5% condition at the 70 and 75 dB SPL levels. The two-factor ANOVA also revealed significant main effects of test time at the 85 and 90 dB SPL stimulus. Post hoc analyses revealed that test time 3 had higher amplitudes than time 2 at 85 and 90 dB SPL. At 30 kHz, there was a three-way interaction. A series of twofactor ANOVAs (test time ⫻ dose) was run at each stimulus level. The only notable outcome was a statistical trend (p ⫽ 0.068) toward a two-way interaction of dose and test time at the 90 dB SPL stimulus level. At 40 kHz, there was only a trend (p ⫽ 0.051) toward a dose ⫻ stimulus level interaction.
Discussion The current study was undertaken to address the issue of whether the duration of isoflurane anesthesia will affect internal, repeated measures studies of the auditory system. If there was a significant impact of isoflurane anesthesia duration, it would render invalid comparisons between any data that were collected at different time points after anesthesia induction. With the significant impact that isoflurane anesthesia can have on OAEs (Ferber-Viart et al, 1998; Cederholm et al, 2012) and auditory evoked potential thresholds (Ruebhausen et al, 2012), the possibility existed that prolonged anesthetic state under isoflurane would lead to significant variations in ABR thresholds, amplitudes, and latencies. Dose of isoflurane has also been shown to have effects on auditory-evoked potential waveforms (Santarelli et al, 2003a,b; Stronks et al, 2010). Therefore, two doses, 1.5% and 2% isoflurane, were examined in the current study to see if the ABRs collected under the two doses differed from each other, and if effects of duration of isoflurane anesthesia were dependent upon dose level. As detailed in the Results, there were no significant effects of duration on thresholds, but there were a number of statistically-significant effects of anesthesia duration (as indexed by the two-way and three-way interactions detailed in the Results) on latency and amplitude of the P1 and P2 waves. Although the differences were often statistically significant, post hoc analyses of the interactions did not show a stable or consistent pattern across the different frequencies tested or test metrics used. The data reported by Cederholm et al (2012) that were obtained from the C57Bl/6J mouse show a consistent effect of reduction in ABR component amplitudes over a 60-minute period of isoflurane anesthesia. Such a consistent effect was not found in the current investigation. The analysis of the large number of variables and testing conditions in the current study was likely to yield some random significant results, and that appears to be the case with the significant effects that were detailed in the Results section. Many of the post hoc analyses of the significant interactions found in the current study resulted in single test times being different from each other at only single stimulus levels, suggesting that the differences were random fluctuations rather than truly impactful findings that would require investigators to review past data collected under isoflurane anesthesia or alter future testing protocols. The current experiment utilized spontaneous breathing during the induction and maintenance of isoflurane anesthesia, as opposed
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to mechanical ventilation as was used in human surgical protocols (Manninen et al, 1985; Ferber-Viart et al, 1998; Buyukkocak et al, 2009) and was used in the Stronks et al (2010) study of the guinea pig. Mechanical ventilation provides a more stable anesthetic level in the respiratory system than spontaneous inhalation does. In conditions of spontaneous breathing, such as in the current study, the induction of anesthetic state can lead to changes in breathing rates. A reduction in breathing rate would subsequently lead to a decrease in isoflurane in the animal’s system. The uncertainty in exposure levels to the isoflurane when delivered through spontaneous breathing could account for some of the variability seen between animals in the current study, as well as the inconsistent effects of the two different doses on the waveform latencies and amplitudes. Whether the difference in outcomes between the current study and the work by Cederholm et al (2012), which also utilized spontaneous breathing, could be due to difference in species or due to the specific differences in protocols used is unknown. Two doses of isoflurane were utilized in the current study, 1.5 and 2%. The expectation was that, if there was any effect of dose, the 2% dose would suppress amplitudes, and possibly prolong latencies, relative to the 1.5% dosing condition. Also, a duration effect seemed more likely to occur in the 2% condition than in the 1.5% condition, since the total amount of inhaled isoflurane would be considerably greater. As was the case with an effect of duration, no consistent significant differences appeared between the two dosing conditions. For example, the effects of dose that were seen in the amplitude measures sometimes saw the 2% condition showing lower amplitudes than the 1.5% condition, but then sometimes showing higher amplitudes than the 1.5% condition. Dose had very few effects on latency, other than reducing the variances between test times for the latencies of P1. The variances were generally lower in the 2% condition, suggesting more stable responses across test times and stimulus levels in comparison with the 1.5% condition. As can be seen in Figures 2–5, the variances are generally smaller for the 2% condition than the 1.5% condition. The most compelling evidence for a significant impact of isoflurane on the ABR was the testing performed at 40 kHz. In the Fischer 344/NHsd rat, 40 kHz is often more variable from animal to animal and test to test in terms of the threshold and ABR input-output functions (Bielefeld et al, 2008a,b), and the Sprague-Dawley appears to be no different. Thresholds were highest at 40 kHz, regardless of test time or dose condition (Figure 1). The error bars for the 40 kHz graphs in Figures 2–5 are consistently larger than other frequencies, indicating greater variability between animals and within the repeated measures for each of the tests. The 40 kHz frequency showed a significant interaction for the P1 and P2 latency measures, and for P1 amplitude. The P1 amplitude effect of isoflurane dose was also among largest differences that affected the highest number of stimulus levels (Figure 3). But like other frequencies and measurements, the effect of isoflurane dose on the P1 amplitude at 40 kHz was not consistent.
Conclusion Although numerous statistically significant effects were detected when comparing duration, dose, and stimulus level at each test frequency, there was no consistent effect on latency or amplitude of the ABR components. Combined with the lack of significant effect on thresholds, the current data indicate that duration of isoflurane anesthesia is not a significant factor for ABR testing in the SpragueDawley rat, at least up to the ~110 minutes of anesthetic state tested
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in the current study. ABR measurements collected within that time frame appear to be equally valid and suitable for comparisons to one another. A longer duration may indeed begin to impact the ABR, as could higher dose levels of isoflurane than those tested in the current study.
Acknowledgements
Int J Audiol Downloaded from informahealthcare.com by University of Windsor on 10/01/14 For personal use only.
The author thanks Ellen Hambley, José Quilles, Katherine Kerns, and Meghan Joyce for their assistance with the data collection and analyses. Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
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