Volatile anesthetics maintain tidal volume and minute ventilation to a greater degree than propofol under spontaneous respiration

Background Although general anesthetics depress spontaneous respiration, the comprehensive effect of general anesthetics on respiratory function remains unclear. We aimed to investigate the effects of general anesthetics on spontaneous respiration in non-intubated mice with different types and doses of general anesthetic. Methods Adult C57BL/6 J mice were administered intravenous anesthetics, including propofol and etomidate, and inhalational anesthetics, including sevoflurane and isoflurane in vivo at doses of 0.5-, 1.0-, and 2.0-times the minimum alveolar concentration (MAC)/median effective dose (ED50) to induce loss of the righting reflex (LORR). Whole-body plethysmography (WBP) was applied to measure parameters of respiration under unrestricted conditions without endotracheal intubation. The alteration in respiratory sensitivity to carbon dioxide (CO2) under general anesthesia was also determined. The following respiratory parameters were continuously recorded during anesthesia or CO2 exposure: respiratory frequency (FR), tidal volume (TV), minute ventilation (MV), expiratory time (TE), inspiratory time (TI), and inspiratory–expiratory time ratio (I/E), and peak inspiratory flow. Results Sub-anesthetic concentrations (0.5 MAC) of sevoflurane or isoflurane increased FR, TV, and MV. With isoflurane and sevoflurane exposure, the CO2-evoked increases in FR, TV, and MV were decreased. Compared with inhalational anesthetics, propofol and etomidate induced respiratory suppression, affecting FR, TV, and MV. In 100% oxygen (O2), FR in the group that received propofol 1.0-times the ED50 was 69.63 ± 33.44 breaths/min compared with 155.68 ± 64.42 breaths/min in the etomidate-treated group. In the same groups, FR was 88.72 ± 34.51 breaths/min and 225.10 ± 59.82 breaths/min, respectively, in 3% CO2 and 144.17 ± 63.25 breaths/min and 197.70 ± 41.93 breaths/min, respectively, in 5% CO2. A higher CO2 sensitivity was found in etomidate-treated mice compared with propofol-treated mice. In addition, propofol induced a greater decrease in FR, MV, and I/E ratio compared with etomidate, sevoflurane, and isoflurane at equivalent doses (all P < 0.05). Conclusions General anesthetics differentially modulate spontaneous breathing in vivo. Volatile anesthetics increase FR, TV, and MV at sub-anesthetic concentrations, while they decrease FR at higher concentrations. Propofol consistently depressed respiratory parameters to a greater degree than etomidate.


Background
More than 300 million major surgical procedures requiring general anesthesia or analgesics are conducted worldwide each year [1,2]. General anesthetics and analgesics induce respiratory depression, which is a critical issue in clinical practice, especially for sedative procedures Open Access *Correspondence: liyu1936@163.com; zhouc@163.com 1 Departments of Anesthesiology, West China Hospital of Sichuan University, Chengdu 610041, China Full list of author information is available at the end of the article requiring maintenance of spontaneous respiration [3]. Multiple studies have explored the depressant effect of opioids on respiration, but the effects of general anesthetics are not well elucidated [4,5].
General anesthetics exert various clinically important actions, including hypnosis, amnesia, and immobility [6], as well as respiratory disturbance. Even though a depressant effect is a commonly suggested effect of general anesthetics, significant differences are observed in respiratory behavior between general anesthetics. Propofol depresses ventilation by affecting central chemoreceptor sensitivity, reducing the ventilatory response to hypercapnia, and reducing the ventilatory adaptation to hypoxia, even at sub-anesthetic doses [7,8]. During dexmedetomidine infusion, respiratory frequency (FR) is significantly increased, and the overall apnea/hypopnea index is significantly decreased [9].
Volatile anesthetics, including isoflurane and sevoflurane, are preferred over intravenous anesthetics, such as propofol and etomidate, in conditions necessitating maintenance of spontaneous respiration, such as anticipated difficulties with endotracheal intubation. Both volatile anesthetics and intravenous anesthetics decrease FR, tidal volume (TV), and minute ventilation (MV) [10][11][12][13]. However, early clinical studies with small sample sizes indicated that volatile anesthetics, including enflurane, isoflurane, and sevoflurane, increase FR [10,14]. Multiple structures associated with respiration are affected by general anesthetics, including the ventral medulla, the retrotrapezoid nucleus (RTN), and phrenic motor neurons [13,15]. Uncovering the respiratoryrelated response in vivo during exposure to general anesthetics may help to identify the underlying mechanism. However, limitations exist in previous studies, including restricted study doses and time courses.
As a physical stimulant, carbon dioxide (CO 2 ) plays an important role in modulation of respiration by general anesthetics. Studies revealed an increase in the partial pressure of CO 2 in artery (PaCO 2 ) after exposure to general anesthetics. The increase in FR under volatile anesthesia is considered to be a compensatory effect resulting from an elevated PaCO 2 and respiratory depression [10,14,16]. However, lack of an increase in FR was found during intravenous anesthesia. Differences in the manipulation of neuronal processes that are sensitive to CO 2 might contribute to the discrepancy in respiratory responses induced by volatile versus intravenous anesthetics. Even so, the effect of general anesthetics on respiratory responses to CO 2 remains unclear.
Inhaled and intravenous anesthesia are two most common approach in clinic. In this study, we chose these four classic drugs of inhaled (sevoflurane and isoflurane) and intravenous anesthetics (propofol and etomidate). The dose-related and time-related effects of the four general anesthetics on respiratory behaviors in mice were explored using whole-body plethysmography (WBP). The respiratory responses to CO 2 during exposure to general anesthetics were also explored. The results of this study may encourage further research into the mechanisms underlying modulation of respiration by general anesthetics.

Animals
All protocols were approved by the Institutional Animal Experimental Ethics Committee of Sichuan University (Chengdu, Sichuan, China) in accordance with the animal care guidelines of the National Institutes of Health. Endeavors were made to minimize suffering and to reduce the number of mice used.
Experiments were performed in wild type C57BL/6 J male mice aged 12 weeks, weighing 20-25 g. All mice were housed in standard conditions, with a 12-h light/ dark cycle and with ad-libitum access to food and water. All experiments were performed during the light cycle (from 9:00 am to 5:00 pm).

Whole-body Plethysmograph
Whole-body Plethysmograph (Buxco FinePointe Series WBP 4-site system, Data Sciences International, New Brighton, MN, USA) offers a precise, non-invasive, quantitative approach to measure respiratory parameters in conscious, freely moving animals. The system relies on a specially designed chamber in which the subject is placed and allowed to breathe freely under natural conditions, unrestrained and untethered.
The volume of the plethysmography chamber was 480 ml. A constant flow was driven by an oxygen (O 2 ) cylinder connected to the chamber, which ensured continuous flow at 0.5 ± 0.1 L/min of gas, thereby preventing CO 2 accumulation. Hyperoxia and hypercapnia and/or volatile anesthetics were continuously induced/ administered into the chamber through the flow pump.
FinePointe software was used to analyze incoming data and create instant reports. The following respiratory parameters were registered: FR, TV, MV, peak inspiratory flow, inspiratory time (Ti), and expiratory time (Te). Except for FR, all measured respiratory parameters (including TV and MV) were normalized to body weight to make them comparable between mice with different body weights. One technician who was blinded to the animal groups measured the respiratory outputs in vivo, and another researcher analyzed the data.
Propofol and etomidate (Fresenius Kabi, Bad Homburg, Germany) were injected i.p. All mice received a similar injected volume to exclude the effects of volume variation on respiratory depression in the propofol and etomidate groups.
For sevoflurane (Abbott Pharmacology Ltd., Co., Shanghai, China) and isoflurane (North Chicago, IL, USA) administration, mice were kept in the plethysmography chamber, which comprised a gas inlet and outlet. Sevoflurane or isoflurane was applied into the chamber through the inlet, with a continuous flow of 100% O 2 at a rate of 0.5 ± 0.1 L/min. Concentrations of sevoflurane and isoflurane were monitored in real-time using the RGM monitor (Datex-Ohmeda, Louisville, CO, USA). For control mice, the chamber was filled with 100% O 2 at a flow rate of 0.5 L/min.
As indicated by our preliminary experiments, the concentration of sevoflurane or isoflurane in the chamber was balanced after 5 min of delivery, which was detected at the outlet. Drug washout was achieved by suctioning the chamber and flowing fresh air into the chamber between each experiment.

Behavior test
On the day of the experiment, mice were transported to the laboratory at least 2 h before the start of the experiment. Prior to recording, mice were placed into the plethysmography chamber with no restriction for a minimum of 2 h to allow acclimatization. Mice were awake and calm during testing. Over-excited mice with a high level of locomotor activity or environmental exploration were excluded from anesthetic treatment and behavioral testing.
All experiments were performed at room temperature (22 °C ± 1.5 °C) and humidity (58% ± 5%). Calibration of the plethysmography chamber system was performed once per day before the experiment according to WBP instructions. After each recording, the chamber was cleaned thoroughly with 75% ethanol.
During the experiments, raw respiratory parameters were continuously recorded, and average values were calculated every 2 s. The behavioral state of mice was classified as immobile, exploring, grooming, or undefined. In practice, only data recorded during immobility was considered in the comparison, at least 15 min after propofol or volatile anesthetic delivery. Treatments and tests were conducted randomly on mice in different groups to preclude potential confounding factors. The behavioral test and respiratory parameter analysis were performed with independent researchers who were blinded to the study aims and protocol.

Statistical analysis
The distribution of values in each set of experiments was tested for normality using the D' Agostino-Pearson omnibus test or the Shapiro-Wilk test. Values are expressed as mean ± standard deviation. The means among groups were compared with with one -way or two-way analysis of variance followed by Bonferroni correction for posthoc analysis. Differences among groups were considered statistically significant when P was < 0.05. Statistical analyses were performed using Prism 7.0 software (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics 22.0 (IBM Corp., NY, USA).

Sub-anesthetic concentrations of volatile anesthetics increase FR, TV, and MV in wild type mice
There were no measurable differences in respiratory activity among all experimental mice when exposed to room air, 100% O 2 , 3% CO 2 , and 5% CO 2 conditions (data not shown). During the comparison of inhalational anesthetics, mice had similar FR, TV, and MV values at baseline (100% O 2 ). Mice showed a reactive increase in FR, TV, and MV when exposed to 1 MAC sevoflurane and isoflurane with 100% O 2 at the beginning. Mice stabilized after 10-12 min of recording. The timeline with 1 MAC of sevoflurane and isoflurane are presented in (Fig. 1 A-F). There was no difference between two groups in FR, TV and MV.

Propofol causes significant respiratory depression compared with etomidate
To explore the dose-dependent effect of intravenous anesthetics on respiratory depression, we used two different concentrations of propofol and etomidate (1 ED 50 and 2 ED 50 , Fig. 4 A-D). We found that compared with 1 ED 50 , propofol depressed TV in the group exposed to 2 ED 50 (P = 0.004) (Fig. 4 Aii). Etomidate also depressed TV at a higher dose (P < 0.001) (Fig. 4 Cii). Scatter plot (tidal volume vs. airflow) also showed that compared to 1 ED 50 , breathing in 2 ED 50 propofol group became slow and less forceful (Fig. 4 Bii). In mice exposed to 1 ED 50 of propofol and etomidate, a time-course study revealed that propofol progressively reduced FR, TV, and MV and stabilized after 20 min during the 30-min exposure (Fig. 5 A, B, C). However, there was no further respiratory depression after respiration decreased to a relatively stable level (after 10 min) in the group that received 1 ED 50 etomidate (Fig. 5 A, B, C). Compared to propofol, etomidate causes less respiratory depression in FR (Fig. 5. D) and MV (Fig. 5 F). In addition, mice that received 1 ED 50 etomidate showed a diminished CO 2 -dependent increase in FR, TV, and MV. However, mice that received 1 ED 50 propofol showed a significant increase in FR from 100% O 2 to 5% CO 2 (Fig. 5. D, P = 0.023), but not TV (Fig. 5. E, P = 0.058) or MV (Fig. 5 F, P = 0.067).

Inhalational anesthetics cause less respiratory depression compared with intravenous anesthetics
Unlike with inhalational anesthetics, mice administered intravenous anesthetics cannot be accommodated in the chamber in the beginning of the experiment. Mice in the propofol and etomidate groups became hyperactive after i.p. injection outside the chamber. Thus, we compared the stable period of each general anesthetics. We found that at equivalent doses (1 MAC or 1 ED 50 ), sevoflurane and isoflurane caused less respiratory depression compared with propofol and etomidate (Fig. 6 A-6  L). Propofol caused significant depression in FR, TV and MV after i.p. injection when compared with sevoflurane and isoflurane (Fig. 6 A-C). In addition, we compared Ti, Te, and Ti/Te ratio among the four anesthetics. All four general anesthetics showed an increase in TE. Among them, propofol decreased the Ti/Te ratio (P < 0.001 when compared with sevoflurane isoflurane and etomidate groups, Fig. 6D) and increased Te from 0.13 ± 0.01 s to 1.00 ± 0.31 s (Fig. 6K).

Discussion
In the present study, we found that sub-anesthetic concentrations of sevoflurane and isoflurane increased FR, TV, and MV. In addition, CO 2 -sensitive respiratory responses were maintained to a greater degree with 0.5 and 1 MAC sevoflurane/isoflurane compared with 2 MAC. Meanwhile, in contrast to the etomidate group, propofol showed a dismissed response to graded increases in CO 2 . A time-course analysis revealed that propofol progressively decreased TI/TE ratio during a 30-min exposure compared with sevoflurane, isoflurane, and etomidate at equivalent doses.
In this study, the respiratory response of adult mice to general anesthetics was measured with WBP. WBP is widely used for precise, non-invasive, quantitative measurement of respiratory parameters in unrestrained conditions without intubation [19,20]. Adult mice were administered sevoflurane, isoflurane, propofol, and etomidate in vivo at doses of 0.5-, 1.0-, and 2.0-times the MAC or ED 50 required to induce LORR, which included the most commonly used dose ranges of inhalational and intravenous anesthetics. In addition, only the state of immobile respiratory parameters was observed with a sufficient duration for subsequent data analysis. The effect of general anesthesia on the CO 2 -sensitive respiratory response was also determined. Concentrations of sevoflurane and isoflurane were monitored in real-time using the RGM monitor to prevent drug and CO 2 accumulation.
General anesthetics modulate ventilation by disturbing central chemoreceptor sensitivity, reducing the  Data are expressed as mean ± standard deviation. *, #, † indicate that there were significant differences compared with sevoflurane, isoflurane, and propofol groups, respectively, by one-way ANOVA (P < 0.05) ventilatory response to hypercapnia, depressing metabolic ventilatory control, and inhibiting the ventilatory adaptation to hypoxia, even at sedative doses [7,8]. In the present study, both sevoflurane and isoflurane preserved spontaneous breathing and the ventilatory response to hypercapnia at sub-anesthetic concentrations. However, higher CO 2 sensitivity was not observed in mice administered 2 MAC sevoflurane/isoflurane. Although multiple sites contribute to the depressive effect of general anesthetics on respiration, the relatively selective maintenance of spontaneous breathing is poorly known. Central CO 2 chemo-sensitivity in mammals is mainly mediated by Phox2B-expressing neurons of the RTN, which were first known for their CO 2 /pH sensitivity and role in providing central chemoreceptor drive to the respiratory system [21][22][23]. Their CO 2 sensitivity is unaffected by pharmacological blockade of the respiratory pattern generator and persists without carotid body input [22]. Volatile anesthetics cause activation of RTN neurons, which serve an important integrative role in maintaining respiratory motor activity under immobilizing anesthetic conditions [24]. Depression by propofol may be attributed to an exclusive effect within the central chemoreflex loop at central chemoreceptors. In contrast to sub-concentrations of inhalational anesthetics, the peripheral chemoreflex loop, when stimulated with CO 2 , remains unaffected by propofol [25].
Previous studies demonstrate that sevoflurane-induced respiratory depression is mediated by medullary respiratory and phrenic motor neurons. γ-Aminobutyric acid type A (GABA A ) receptors may be involved in sevoflurane-induced respiratory depression within the medulla, but not within the spinal cord [13,26]. Many neuronal elements within the respiratory system are inhibited by inhalational anesthetics. The mammalian pre-Bötzinger complex is an excitatory network of neurons in the medulla that is critically involved in respiration [27]. The effect of inhalational anesthetics on TASK-like channels plays a major functional role in chemosensory modulation of respiratory rhythm in the pre-Bötzinger complex [28]. However, these reported mechanisms have not yet elucidated the difference between the effects of isoflurane and sevoflurane.
In the present study, propofol displayed more obvious respiratory depression on FR, TV, and MV compared with etomidate and volatile anesthetics. Propofol at 1 ED50 exhibited rapid and significant respiratory depression approximately 3 min after i.p. injection compared with 1 ED50 etomidate in our study. Propofol also induced the greatest decrease in TI/TE ratio among the four general anesthetics. Propofol may cause significant airway obstruction [29]. Sedative doses of propofol cause a phase shift between abdominal and ribcage movements under spontaneous breathing without airway support (like the WBP method in our study), thereby decreasing the contribution of ribcage movement to TV and disturbing arterial oxygen tension [30]. It is likely that GABA A receptor-mediated hyperpolarization of neurons serves as the neuronal basis of propofol-induced respiratory depression in vivo [31,32]. Our study provides new insight into the effect of general anesthetics on respiratory behavior, but further study is needed to uncover the underlying mechanisms. One limitation of our study is that we did not include other types of general anesthetic and analgesics, such as ketamine and opioids.

Conclusion
In conclusion, the present study systematically investigated modulation of respiratory function by general anesthetics and revealed that sevoflurane and isoflurane increase respiratory parameters, even at sub-anesthetic concentrations. Propofol and etomidate depressed TV, but not FR, at higher doses. In addition, propofol induced the greatest decrease in TI/TE ratio among the four general anesthetics. These results suggest that in cases requiring maintenance of spontaneous respiration, sevoflurane or isoflurane may be a better choice than propofol or etomidate.