We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.
Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under “physiologic” conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled “air” fractions and “medium” concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure.
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.
Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by diffuse alveolar injury, lung edema, and hypoxemic respiratory failure. Although ARDS represents more than 10% of intensive care unit (ICU) admissions and nearly 25% of ICU patients requiring mechanical ventilation, it is still an under-recognized challenge for clinicians, with a hospital mortality rate of 35-45%1. Despite intense research, the identification of an effective ARDS pharmacologic therapy or prevention has failed to date. Two major features contribute to mortality in ARDS: impaired alveolar fluid clearance (AFC) (i.e., the altered resorption of alveolar edema fluid from distal lung airspaces) and severe inflammation2. Since ARDS mortality remains high, current initiatives should also include primary prevention; however, a key challenge is to identify at-risk patients in whom ARDS is likely to develop and who would benefit if ARDS were prevented.
Volatile halogenated anesthetics, such as sevoflurane and isoflurane, are widely used to provide general anesthesia in the operating room. Worldwide, more than 230 million patients undergoing major surgery each year require general anesthesia and mechanical ventilation3, and postoperative pulmonary complications adversely affect clinical outcomes and healthcare utilization4. The use of sevoflurane instead of propofol was associated with improved lung inflammation in patients undergoing thoracic surgery and significant decreases in adverse events, such as ARDS and postoperative pulmonary complications5. Similarly, pretreatment with isoflurane had protective effects on respiratory mechanics, oxygenation, and hemodynamics in experimental animal models of ARDS6,7. Although further studies are warranted to address the impact of inhaled agents on outcomes in noncardiac surgery, a similar decrease in pulmonary complications has been recently observed in a meta-analysis, demonstrating that inhaled anesthetic agents—as opposed to intravenous anesthesia—are significantly associated with a reduction in mortality for cardiac surgery8.
Specific prospective data about the use of volatile agents for the sedation of ICU patients to prevent or treat lung damage is lacking. However, several trials now support the efficacy and safety of inhaled sevoflurane for the sedation of ICU patients, and preclinical studies have shown that inhaled sevoflurane and isoflurane7,9 improve gas exchange, reduce alveolar edema, and attenuate inflammation in experimental models of ARDS. Additionally, sevoflurane mitigates type II epithelial cell damage10, whereas isoflurane maintains the integrity of the alveolar-capillary barrier through modulation of tight junction protein11. However, further studies are needed to verify to what extent the experimental evidence of organ protection from inhaled sevoflurane and isoflurane could be translated to humans. A first single-center randomized controlled-trial (RCT) from our group found that early use of inhaled sevoflurane in patients with ARDS was associated with improved oxygenation, reduced levels of some pro-inflammatory markers, and reduced lung epithelial damage, as assessed by the levels of the soluble form of the receptor for advanced glycation end-products (sRAGE) in plasma and alveolar fluid12.
Taken together, the beneficial effects of sevoflurane and isoflurane on lung injury could point to multiple biological pathways or functional processes that are dependent on the RAGE pathway, namely alveolar fluid clearance (AFC), epithelial injury, translocation of nuclear factor (NF)-κB, and macrophage activation. In addition, sevoflurane may influence the expression of the RAGE protein itself. Since previous research by our research team and others supports pivotal roles for RAGE in alveolar inflammation and lung epithelial injury/repair during ARDS, we designed an experimental model to provide a translational understanding of the mechanisms of sevoflurane in lung injury and repair13,14,15. The in vitro effects of sevoflurane and isoflurane were investigated in a novel human alveolar epithelial primary cell line specifically designed to study the air-blood barrier of the peripheral lung, hAELVi (human Alveolar Epithelial LentiVirus immortalized), with alveolar type I-like characteristics including functional tight junctions16.
While preparing the design of our in vitro investigations (e.g., cultures of alveolar epithelial cells at the air-liquid interface with exposure to "inhaled" sevoflurane or isoflurane, we understood from previously published studies that fractions of sevoflurane have only been assessed in the "air" interface17,18,19 using standard monitors (similar to those used in a clinical setting). Halogenated agent concentrations were usually chosen according to the minimum alveolar concentration (MAC) values (e.g., in humans, for sevoflurane, 0.5, 1.1, and 2.2 vol%, representing 0.25, 0.5, and 1 MAC, respectively; for isoflurane, 0.6, 0.8, and 1.3 vol% representing 0.25, 0.5, and 1 MAC, respectively)20. Indeed, sevoflurane and isoflurane concentrations have never been investigated in the culture medium itself, thus limiting the validity of previous experimental models/instruments. Furthermore, most experiments used an anaerobic jar that was sealed after the air mix containing sevoflurane had been flushed inside. As our goal was to study alveolar epithelial cells under "physiologic" conditions, we believed that such an anaerobic state may not be optimal and would not be compatible with long experimental durations. Therefore, we developed our own system to culture cells at the air-liquid interface and expose them to halogenated agents (sevoflurane and isoflurane) with the aim of providing precise controlled "air" fractions and "medium" concentrations for these agents. In our opinion, this experimental step, which has not been reported to date in the literature, is mandatory prior to any further in vitro investigations of sevoflurane and isoflurane.
1. Culture of Alveolar Epithelial Cells (hAELVi)
2. Preparation of an Air-tight Chamber
NOTE: The construction plan for the air-tight chamber is depicted in Figure 1.
3. Expose Alveolar Epithelial Cells to Halogenated Agents (Sevoflurane and Isoflurane)
NOTE: A schematic drawing of the device is depicted in Figure 2.
CAUTION: Although animal studies have revealed no evidence of fetal harm or impaired fertility, and a very small study during cesarean sections did not show any untoward effects on the mother or fetus, the safety of using halogenated agents (e.g., sevoflurane or isoflurane) during labor and delivery has not been demonstrated to date. Furthermore, no controlled data has been collected during human pregnancies. Therefore, performing experiments using sevoflurane or isoflurane while pregnant should be strongly discouraged.
4. Measure Sevoflurane or Isoflurane by Chromatography
The concentrations of the sevoflurane and isoflurane, which dissolved in the medium over time, are reported in Table 1 and Table 2, respectively.
The courses of the sevoflurane and isoflurane concentrations in the medium were similar over time. Immediately after the required concentration of halogenated agent was set, concentrations rose over the first hour. A plateau was then reached, which persisted until the administration of the halogenated agent was stopped. After administration interruption, concentrations decreased within one hour (Figure 3).
After the first hour, the median concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. No significant difference was found between the different experiments.
Figure 1: Construction plan of the air-tight chamber Please click here to view a larger version of this figure.
Figure 2: Schematic drawing of the device Please click here to view a larger version of this figure.
Figure 3: Concentration of sevoflurane (n = 5) and isoflurane (n = 5) over time. A) Concentration of halogenated agent over time. Values are expressed in mg/L. Values are expressed in mean and SEM. B) Concentration of halogenated agent over time for each experiment. Value are expressed in mg/L. C) Fraction of halogenated agent over time in the air-tight chamber measured by the gas analyzer. Values are expressed in percentages. Please click here to view a larger version of this figure.
Time | Concentration of sevoflurane in the medium | Fraction of sevoflurane | |
(mg/L) | in the air-tight chamber | ||
Median | IC | (%) | |
5 min | 27 | [19 – 31] | 4.6 |
30 min | 152 | [142 – 152] | 4.1 |
1 h | 251 | [243 – 332] | 4.1 |
4 h | 259 | [256 – 271] | 4.2 |
8h | 265 | [237 – 280] | 4.2 |
24 h (stop) | 218 | [196 – 247] | 4.3 |
Stop + 5 min | 237 | [214 – 241] | 0 |
Stop + 30 min | 92 | [91 – 104] | 0 |
Stop + 1 h | 57 | [42 – 58] | 0 |
Table 1: Concentrations of sevoflurane dissolved in the medium over time. Numerical data are expressed as a median value with interquartile range for the concentration and as percentage for the fraction. IQR (for interquartile range)
Time | Concentration of isoflurane in the medium | Fraction of isoflurane | |
(mg/L) | in the air-tight chamber | ||
Median | IC | (%) | |
5min | 2 | [2 – 2.5] | 0.8 |
30min | 16 | [4 – 18] | 1.3 |
1h | 22 | [18 – 27] | 0.9 |
4h | 30 | [25 – 31] | 1.4 |
8h | 22 | [15 – 26] | 1.2 |
24h (stop) | 26 | [23 – 27] | 1.1 |
Stop + 5min | 19 | [12 – 25] | 0 |
Stop + 30min | 4 | [4 – 4] | 0 |
Stop + 1h | 1 | [0.8 – 1] | 0 |
Table 2: Concentrations of isoflurane dissolved in the medium over time. Numerical data are expressed as a median value with interquartile range for the concentration and as percentage for the fraction. IQR (for interquartile range)
Our protocol describes an easy method to expose cells to a precise fraction of a halogenated anesthetic agent, such as sevoflurane or isoflurane. Furthermore, we report here—for the first time—a rigorous correlation between both the gas fraction and the concentration of sevoflurane and isoflurane inside the culture medium itself. This fundamental step now allows us to safely use our air-tight chamber to study the effects of these halogenated agents in a cultured monolayer of human alveolar epithelial cells.
Currently, most research teams studying the effects of sevoflurane in alveolar cells use a jar that is first saturated with halogenated gas and then sealed. In this case, sevoflurane may be metabolized, and it could be speculated that the fraction of volatile agent may decrease linearly over time, leading to an unstable gas concentration. However, the correlation between the gas fraction of sevoflurane and its concentration in the culture medium is not clearly reported in the literature. Usually, the concentration of sevoflurane used in these experiments is chosen based on a simple relationship between the gas fraction and the MAC. MAC was introduced in 1965 and is the concentration of a vapor in the lungs that is needed to prevent a motor response (movement) in 50% of subjects in response to a surgical stimulus (pain)22. MAC is used to compare the strength, or potency, of anesthetic vapors. In ICU patients, MAC is correlated to FeSevo and the clinical Richmond Assessment Agitation-Sedation Scale (RASS)23. Although it is a useful indicator in daily clinical practice, the relevance of this parameter has never been investigated in the setting of experimental in vitro research. In our protocol, using chromatography analyses of the medium, we determined the precise correlation between the sevoflurane contained in the gas fraction and the sevoflurane dissolved into the medium. With this method, the specific effect of a volatile agent is expressed according to the real concentration in the medium rather than based on the approximation of a clinical effect. This important element allows the study of the specific effect of a precise concentration of a halogenated agent on cells growing in a medium, in order to compare the effects of different concentrations of inhaled agents. Furthermore, as the air-tight chamber is very easy to use, this method allows researchers to replicate the experiment with precision.
Another important point that may preclude the use of the correlation between gas fraction and MAC in experimental research is that a halogenated agent has low solubility in blood (blood/gas partition coefficient at 37°C = 0.63 to 0.69 for sevoflurane). A minimal quantity of sevoflurane is mandated to dissolve in the blood before the pressure in the alveoli achieves equilibrium with the pressure in the arterial. Thus, during the induction of anesthesia, the alveolar (end-tidal) concentration (AF, alveolar fraction) of sevoflurane rapidly increases around the inspired concentration (FI, inspired fraction). However, in vitro culture conditions do not allow such mechanisms, and usual cell media mainly consist of aqueous solutions. Furthermore, the solubility coefficient between water/gas (partition coefficient at 37°C = 0.36 for sevoflurane) is lower than between blood and gas, underlying the critical importance of performing chromatography analyses.
Additionally, when a sealed jar is used, the atmospheric oxygen in the jar is absorbed by the cells with the simultaneous generation of carbon dioxide. This effect is probably insignificant in short experimental procedures, but for longer experimental durations, cells that are deprived of oxygen would switch to an anaerobic metabolism; this change in metabolism may induce a certain degree of bias in experimental analyses. In contrast to the sealed jar, when using our air-tight chamber, both oxygen and halogenated agent flows are adjustable over time to maintain the targeted level. This major characteristic of our protocol therefore allows the design of in vitro experiments for long time periods (e.g., more than one day), making it an interesting tool to study the cellular mechanisms involved in lung epithelial injury and repair over time, especially when halogenated agents are used. Indeed, the effects of inhaled anesthetic agents on lung cells or tissue during alveolar injury remain poorly investigated to date while this alternative therapy seems to show very encouraging results12.
However, there are limitations to this technique. First, an anesthetic machine circuit is needed to provide oxygen, carbon dioxide, and halogenated agent gas flows. Using such a device is mandatory to set the flow rate and maintain stable concentrations over time. Second, to sample the medium prior to chromatography analyses, the air-tight chamber is briefly opened, which induces a transient decrease in the gas concentrations. As we use an anesthetic machine circuit, gas flows are thereafter increased until expected concentrations are achieved again on the gas analyzer. Third, we have measured the concentration in the medium for only one fraction of each halogenated agent, chosen a priori based on previous study. Fourth, to stabilize the cell medium to the growth of alveolar epithelial cells, we need to use carbon dioxide at a concentration of 5%. Indeed, no anesthetic machine circuit provides such a concentration of carbon dioxide. Therefore, the anesthetic machine circuit needs to be customized to allow the connection of carbon dioxide gas flow in place of nitrous oxide. Such a connection should be used exclusively in the setting of experimental research and should cautiously be unplugged after each experiment. Furthermore, to avoid any risk for humans, we invite researchers to use a devoted anesthetic machine circuit to perform this protocol and not to use a machine dedicated to clinical anesthesia.
The main advantages of this technique are that it is relatively inexpensive and very easy to adopt, even when researchers have never manipulated an airtight chamber before. Moreover, with our protocol, the results of dissolved sevoflurane and isoflurane concentrations are reproducible, which represents a major quality criterion for experimental research. In addition, our system could allow for the study of other volatile halogenated agents, such as desflurane. Indeed, a simple change of the type of gas evaporator device would be sufficient in this case. Similarly, our system could provide a means to study the concentrations of sevoflurane or isoflurane dissolved in any type of medium with different solubilities, such as water, blood, or oil.
Our experiment represents a fundamental step that is part of a larger project designed to test the hypothesis that sevoflurane and isoflurane may exert beneficial effects on lung injury, inflammation, and AFC through RAGE-mediated pathways. A primary culture of human alveolar epithelial cells will be used for mechanistic investigations of transepithelial fluid transport, channel-specific fluid transport (e.g., using pharmacological antagonism), epithelial paracellular permeability, wound repair, cell migration and proliferation, with or without a halogenated anesthetic agent (sevoflurane or isoflurane), alone or combined with cytomix (in vitro model of alveolar injury)24.
In conclusion, this protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for this frequent and life-threatening syndrome.
The authors have nothing to disclose.
The authors acknowledge the Auvergne Regional Council ("Programme Nouveau Chercheur de la Région Auvergne" 2013) and the French Agence Nationale de la Recherche and the Direction Générale de L'Offre de Soins ("Programme de Recherche Translationnelle en Santé" ANR-13-PRTS-0010) for the grants. The funders had no influence in the study design, conduct, and analysis or in the preparation of this article.
Sevoflurane | Baxter | Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged | |
Isoflurane | Virbac | Performing experiments using sevoflurane or isoflurane while being pregnant should be strongly discouraged | |
Human Alveolar Epithelial cells | InScreenex | INS-CI-1015 | |
huAEC Medium (ready-to-use) | InScreenex | INS-ME-1013-500ml | |
Anesthetic machine circuit | Drager | Fabius | |
Gas analyzer | Drageer | Vamos Plus | |
Anesthetic gas filter | SedanaMedical | FlurAbsord | |
Heated Humifier | Fisher&Paykel | MR850 | |
Chamber | Curver | 00012-416-00 | |
Gas chromatography coupled with mass detection | Thermo Fisher Scientific, San Jose, CA, USA | Trace 1310 with TSQ 8000evo | |
Fused-silica column (30 m x 1.4 µm, 0.25 mm ID) | Restek, Lisses, France | Rxi-624Sil MS |