The requirements of anesthetics for the induction of the anesthetic state vary depending on age, gender, race, and the condition of the patient.1–4 It is possible that the gravitational environment may also alter the effect of anesthetics. In 1997, the United States and Russia conducted a space mission called “Bion 11,” in which 2 macaque monkeys flew in space for 14 days. Although there were plans to collect muscle samples under general anesthesia on the first postflight day, one of the monkeys died during anesthesia and the other suffered from serious complications after anesthesia.5 The most prominent environmental feature during spaceflight is microgravity, a condition in which there is very little net gravitational force, which induces multiple physical changes. Although these physical changes are assumed to alter the effects of anesthetics,5,6 no study has yet reported on the influence of the gravitational environment on the effects of anesthetics.
Chronic exposure to microgravity induces muscle atrophy, decrease in bone volume, changes in body composition, cardiovascular deconditioning, and plastic alteration of the vestibular system (Table 1).7,9,10,13,14,16 Interestingly, some of these physical changes are also seen in hypergravity, even though gravity moves from 1g in a different direction. Decreases in body fluid, lean body mass, and suppressed body mass gain were reported both in hypergravity and microgravity.7–9 Because changes in body fluid and body mass may directly affect the distribution of anesthetics, both hypergravity and microgravity environments presumably alter the effect of anesthetics. Moreover, plastic alteration of the vestibular system and impairment of cardiovascular regulation were also reported in both microgravity and hypergravity.13–16 Because vestibular inputs project to multiple parts of the brain,17 alteration of the vestibular system may affect the pharmacodynamics of anesthetics.
In the present study, we hypothesized that hypergravity may alter the anesthetic effect through an alteration in the distribution of the anesthetic or vestibular-mediated pathway. To investigate this hypothesis, we used the IV anesthetic, propofol, and compared its effects on rats raised in a 1g and a 3g environment for 14 days by measuring the induction of burst suppression in the electroencephalograms (EEGs), arterial blood pressure, and the appearance of the righting response to noxious electrical stimulations. Plasma propofol concentrations were measured to examine the distribution of propofol. To examine whether the vestibular system participated in the observed results, experiments were also conducted on rats with vestibular lesions (VLs).
The animals used in this study were maintained in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science from the Physiological Society of Japan. Experiments were approved by the Animal Research Committee of Gifu University. Male Sprague-Dawley rats (Japan SLC Inc., Shizuoka, Japan) weighing 318 to 398 g (14 weeks old, n = 48) were used.
Five days before exposure to hypergravity, we performed VL procedures on 24 of the 48 rats and sham procedures on the remaining 24 rats under light sedation with isoflurane (Escain; Mylan, Osaka, Japan). Sodium arsanilate solution (100 mg/mL) or saline was injected into the bilateral middle-ear cavity (50 μL/ear) for VLs and sham, respectively. The success of VLs was confirmed by observing the swimming behavior of the rats after they were gently placed in a small tub of water. Rats with complete VLs were unable to determine the direction in which they had to swim to reach the surface and continued to turn around under water.
Rats were assigned to 4 groups: 1G-sham (sham-treated rats continuously raised in a 1g environment, n = 12), 1G-VL (rats with VLs continuously raised in a 1g environment, n = 12), 3G-sham (sham-treated rats raised in a 3g environment for 14 days, n = 12), and 3G-VL (rats with VLs raised in a 3g environment for 14 days, n = 12).
From each group, 6 rats were prepared to examine the effects of propofol. These rats had EEG electrodes placed in them under isoflurane anesthesia 3 days before hypergravity exposure. EEG electrodes consisted of stainless steel screws and a 4-cm lead wire. After exposing the skull, 3 burr holes were opened at the bilateral parietal bones (±5 mm lateral and 5 mm backward from the bregma) and the occipital bone (13 mm backward from the bregma), and the screws were placed on the epidural space. The screws were tightly fixed to the skull with dental resin, and the lead wires were sewed to the back of the neck. Antibiotics (penicillin G [5000 U]; Meijiseika, Tokyo, Japan) and buprenorphine (Lepetan [3 μg/kg]; Otsuka Pharmaceutical Co., Tokyo, Japan) were administered for 3 days after the operation.
The 3g environment was induced by centrifugation (Shimadzu, Kyoto, Japan). Rats were raised in individual cages, which were set in a custom-made gondola-type rotating box for 14 days. During a daily 30-minute break, the cages, feedboxes, and water bottles were refreshed. All rats had access to food and water ad libitum. Room temperature was maintained at 24°C with a 12-hour light/dark cycle. Three days before the experiment (11th day of hypergravity exposure), polyethylene catheters (Intramedic PE50; Becton Dickinson, Franklin Lakes, NJ) were inserted into the femoral artery and vein under isoflurane anesthesia for arterial blood pressure measurement, blood sampling, and propofol infusion. The tips were plugged and sewed to the back of the neck through the subcutaneous tissue. Antibiotics were administered after the operation.
On the day of the experiment, rats were brought out of 3g and rested in 1g for 1 to 2 hours. Control rats were prepared in the same procedure except that they were continuously raised in 1g. Arterial blood (200 μL) was withdrawn to measure hematocrit and plasma albumin concentrations. Removed blood was replaced with the same volume of saline. For arterial blood pressure measurement, an arterial catheter was connected to a pressure transducer (MP5200, Baxter, Deerfield, IL) fixed to the cage at the heart level. The transducer was connected to an amplifier (AP-641G, Nihon Kohden, Tokyo, Japan). EEG was recorded between left parietal and occipital electrodes. The right parietal electrode was used as a reference. Electrodes were connected to an amplifier (MEG-1200, Nihon Kohden) via a head amplifier (JB-101J, Nihon Kohden), with low cut filter 0.5 Hz and high cut filter 100 Hz. Amplified arterial blood pressure and EEG signals were continuously recorded using an analog–digital converter (PowerLab; ADInstruments, Dunedin, New Zealand) at a sampling rate of 200 Hz and 1 kHz, respectively. Heart rate was calculated from the arterial pressure waveform. After baseline arterial blood pressure and EEG recordings, 1% propofol (Maruishi, Osaka, Japan) 20 mg/kg was infused IV for 5 minutes. The time it took for the appearance of the burst suppression with longer than a 2-second suppressive duration in the EEG was used as an indicator of the propofol requirement needed to obtain a deep anesthetic state. Two minutes after the termination of propofol infusion, rats were placed in a supine position and 2 needle electrodes were attached to the right foot. Then, electrical stimulations of 20 Hz and 2 mA were applied for 1 second every 1 minute (NS-101, Unique Medical, Tokyo, Japan). The time it took for the appearance of the righting response induced by the noxious electrical stimulations was used as an indicator of the emergence from propofol anesthesia. In addition, a 30-second averaged arterial blood pressure was automatically calculated using a macro of LabChart (version 7.3.7, ADInstruments) every 1 minute. The time course of mean arterial blood pressure and its nadir after the termination of propofol infusion were used as indicators of the anesthetic state. Rectal temperature was monitored and kept at approximately 37°C using a heating ramp.
The remaining 6 rats in each group were used for measuring plasma propofol concentrations. On the day of the experiment, propofol was infused by the same procedure as described above (20 mg/kg for 5 minutes). Arterial blood samples (200 μL) were withdrawn at the termination of propofol infusion and at 3, 6, and 9 minutes thereafter. To prevent dilution, sampled blood was not replaced with any other fluid. The blood samples were immediately centrifuged (6000g, 15 minutes) and stored at −80°C for later analysis. Propofol concentrations of the plasma were analyzed using high-performance liquid chromatography (LC-10AVP; Shimadzu).
In the present study, 6 rats per group were used for the evaluation of propofol effects and plasma propofol concentration, respectively (total 48 rats). All rats successfully completed the examination. The sample size was determined by the preliminary experiments, which had a primary outcome of the time for the righting response to appear. The righting response in 3G-sham rats (25 ± 1 minute) appeared 15 minutes later than it appeared in the 1G-sham rats (40 ± 9 minutes). Thus, we calculated the sample size (N) with the following equation, with estimated SD = 9, Δ = 15 and set α = 0.05, β = 0.20.
N = 2 × (Zα/2 + Zβ) × SD2/Δ2 = 2 × (1.96 + 0.84)2 × 92/152 = 5.64
where N = sample size, α = α error, β = β error, Zα/2 = point of upper α/2 in normal distribution, Zβ = point of upper β in normal distribution, SD = standard deviation, and Δ = difference of mean. Thus, 6 rats per group were used in the present study.
All data are presented as means ± SD. Statistical analysis was performed by Ekuseru-toukei 2012 (Social Survey Research Information Co. Ltd., Tokyo, Japan) and R (version 3.0.1, R Foundation for Statistical Computing, Vienna, Austria). Before all statistical analyses, the Shapiro–Wilk test and Bartlett test were performed to assess normal distribution and equal variances, respectively. Analysis for baseline data, mean time taken for the appearance of burst suppression, the nadir of mean arterial blood pressure, and mean time taken for the appearance of the righting response were performed by 1-way analysis of variance (ANOVA). The time courses of mean arterial blood pressure and plasma propofol concentrations were compared by repeated-measures 2-way ANOVA. After 1-way or 2-way ANOVA, if the F ratio indicated statistical significance, the Tukey–Kramer post hoc test was used for among-group comparisons. P < 0.05 was considered statistically significant.
Table 2 shows baseline data obtained just before propofol infusion. In rats raised in the 3g environment, body weights were significantly lower compared with rats raised in the 1g environment. VL had no significant effect on the body weight of rats raised in the 1g environment. However, VL significantly ameliorated the 3g-induced body weight loss. There were no differences between groups in baseline mean arterial blood pressure, heart rate, hematocrit, and plasma albumin concentration.
Representative recordings of arterial blood pressure, EEG, and electrical stimulation are shown in Figure 1. Arterial blood pressure gradually decreased with propofol infusion; the nadir of arterial blood pressure was seen at the end of the infusion. The nadir of mean arterial blood pressure was lower in 3G-sham rats (61.8 mm Hg) than in 1G-sham rats (91.0 mm Hg). Along with the propofol infusion, the EEG changed into high-voltage slow wave (Figure 1B), then to burst suppression (Figure 1C). To examine the time taken for the appearance of the righting response, electrical stimulations were applied. In a 1G-sham rat, the righting response was observed at 20 minutes after the termination of propofol infusion. This duration was prolonged in a 3G-sham rat (35 minutes).
Induction and emergence of anesthesia are summarized in Figure 2. In 3G-sham rats, earlier induction of burst suppression in the EEG and later appearance of the righting response than other groups were observed in all rats (upper panel). Lower panels show the comparison of each outcome among groups. The mean time for burst suppression to appear was significantly earlier in 3G-sham rats than 1G-sham rats (lower left panel, 95% confidence interval [CI], 31.5–111.8 seconds, P = 0.00037). The mean time taken for the appearance of the righting response was significantly postponed in 3G-sham rats (lower right panel, 95% CI, 8.8–25.2 minutes, P < 0.0001 vs 1G-sham). Both of these effects were not observed in 3G-VL rats (mean time for burst suppression to appear: 95% CI, –32.5 to 47.8 seconds, P = 0.95; mean time for the righting response to appear: 95% CI, –6.9 to 9.2 minutes, P = 0.98 vs 1G-sham); whereas, VL itself had no effects on mean time for burst suppression to appear (95% CI, –47.8 to 32.5 seconds, P = 0.95 vs 1G-sham) or mean time for the righting response to appear (95% CI, –5.2 to 10.9 minutes, P = 0.76 vs 1G-sham) if rats were raised in 1g environment.
Figure 3 shows the time course of mean arterial blood pressure (upper panel) and its nadir (lower panel). Along with the propofol infusion, mean arterial blood pressure decreased and reached nadir in 1 to 2 minutes after the termination of propofol infusion and then gradually recovered toward preinfusion levels. Mean arterial blood pressure tended to be lower in 3G-sham rats, but it did not reach a significant level (the largest difference of mean arterial blood pressure between 1G-sham and 3G-sham rats was observed at 11 minutes after the termination of propofol infusion; 95% CI, –5.6 to 44.6 mm Hg, P = 0.17). On the contrary, the nadir of mean arterial blood pressure was significantly lower in 3G-sham rats (95% CI, 3.6–47.5 mm Hg, P = 0.019 vs 1G-sham). This effect was abolished by VLs (95% CI, –13.8 to 30.2 mm Hg, P = 0.73 vs 1G-sham).
Figure 4 shows the time course of mean plasma propofol concentrations at 0, 3, 6, and 9 minutes after the termination of propofol infusion. In all groups, mean plasma propofol concentrations were highest just after the propofol infusion and gradually decreased in 9 minutes. There was no difference in mean plasma propofol concentrations among groups at each time point (the F ratio did not indicate statistical significance, P = 0.67).
The major findings of this study are as follows: In 3G-sham rats, the mean time taken to induce burst suppression in the EEG was earlier and the nadir of mean arterial blood pressure after the termination of propofol infusion was lower compared with 1G-sham rats, and mean time taken for the appearance of the righting response to noxious electrical stimulations was prolonged. These changes were abolished by VLs. However, there was no difference among groups in mean plasma propofol concentrations at 0, 3, 6, and 9 minutes after the termination of propofol infusion.
The effects of propofol cannot be defined by a single indicator because anesthetic drugs, including propofol, have multiple clinical outcomes, such as hypnosis, amnesia, immobility, and circulatory dysfunction.18,19 We used multiple indicators to examine the effects of propofol. These included the time taken to induce burst suppression in the EEG, the nadir of mean arterial blood pressure after the termination of propofol infusion, and the time taken for the appearance of the righting response to noxious electrical stimulations. Burst suppression in an EEG is a reliable indicator of deep anesthesia. It is reported that if burst suppression appears, 95% of the cortical cells are silent during suppression and cerebral metabolism is greatly suppressed, that is, a condition of deep anesthesia.20–22 In this study, burst suppression appeared earlier in the 3G-sham group than in the other groups, suggesting that the 3G-sham rats required smaller amounts of propofol to induce a deep anesthetic state.
Because propofol suppresses the vasomotor center to decrease sympathetic nerve activity and directly acts on vascular smooth muscle to decrease its tonus, propofol induces hypotension.23–25 We compared the nadir of mean arterial blood pressure after propofol infusion as an indicator of the cardiovascular effect of propofol. In 3G-sham rats, the nadir of mean arterial blood pressure was significantly lower. This result suggests that the cardiovascular effect of propofol was increased in 3G-sham rats. Together with earlier induction of burst suppression in the EEG, 3G-sham rats were considered to be in a deeper anesthetic state at the termination of propofol infusion, even though the same dose of propofol (20 mg/kg) was infused in all groups.
To examine the duration of the effect of propofol, we measured the time taken for the appearance of righting responses to noxious electrical stimulation. The appearance of the righting response indicates that the plasma propofol concentration is lower than the threshold required for the anesthetic state, especially immobility. The 3G-sham group had a significantly longer anesthetic state than the other groups. This result indicates that the propofol concentration might have remained higher in 3G-sham rats, or alternatively, the propofol sensitivity of the central nervous system (CNS) might have increased in 3G-sham rats.
To examine whether the increased propofol effect was caused by higher plasma propofol concentrations, we examined the time course of plasma propofol concentrations. Because the propofol infusion dose was normalized by total body weight, changes in body composition could have altered distribution and plasma concentrations of the propofol.4,26 There was no difference in mean plasma propofol concentrations at each time point among the groups, whereas the propofol effects were increased in the 3G-sham group. These results indicate that the increased effects of propofol in 3G-sham rats were not brought about by higher plasma propofol concentrations. We also confirmed that there were no differences in hematocrit and plasma albumin levels among the groups. Plasma albumin can affect the propofol effect because propofol extensively binds to albumin and effective “free propofol” concentrations can be changed depending on the plasma albumin concentration.27 Therefore, our results suggest that the increased effects of propofol observed in 3G-sham rats were not brought about by the alteration of free propofol levels. These results also suggest that blood volume proportion to body weight was the same in all 4 groups. This idea is consistent with a previous report that suggested that the ratio of body fluid to body mass was not changed after 2g exposure for 12 days, although significant decreases in total body water were observed.8
In the present study, we used 2 groups of rats to examine propofol effects and to measure plasma propofol concentration. Because arterial blood pressure was used as an indicator of anesthetic depth, hemodynamic effect of repeated blood sampling should have been avoided. If withdrawn blood is replaced by other fluids, the hemodynamic effect of withdrawn blood might be minimized; however, the propofol concentration might be affected. Accordingly, the pharmacokinetics and pharmacodynamics of propofol could not be evaluated simultaneously. This is a limitation of the present study.
The results of the present study suggest that the vestibular system is involved in the increased effects of propofol. Gravitational input is sensed by the vestibular system and put into the vestibular nucleus complex, from which it projects to multiple sites, including the oculomotor apparatus, spinal cord, inferior olive, thalamus, hypothalamus, reticular formation, and cortex.17 Many changes in the CNS have been reported under different gravitational environments; for example, the downregulation of dopamine D2 receptors and upregulation of serotonin 5HT-1 receptors by microgravity,28 increase of serotonin in cerebrospinal fluid by hypergravity,29 reduction in mRNA expression of glutamate Glu R2 receptors by hypergravity,30 and reduction of γ-aminobutyric acid (GABA) immunoreactivity in axon terminals by hypergravity.31 Because propofol directly and indirectly activates GABA type A (GABAA) receptors and participates in the reduction of neural activity,32 it is thought that if vestibular-mediated CNS alteration includes GABAA receptors, it can alter the effects of propofol. Future studies are required to clarify the mechanism of increased propofol sensitivity in CNS.
In conclusion, the present study provides evidence that hypergravity induced an increased effect of propofol which was not caused by differences in plasma propofol concentrations, but by a vestibular-mediated pathway.
Name: Chihiro Iwata, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Chihiro Iwata has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Chikara Abe, DDS, PhD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Chikara Abe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Mitsuhiro Nakamura, PhD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Mitsuhiro Nakamura has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Hironobu Morita, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Hironobu Morita has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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