Several volatile general anesthetics exert irritant side effects in the airways when inhaled. The pungent volatile anesthetics isoflurane and desflurane can induce coughing and bronchospasm and laryngospasm during induction of anesthesia as a result of C-fiber activation.1 It has been shown that pungent general anesthetics, including propofol, interact with members of the transient receptor potential family (TRP), activating the nearly universal chemosensor TRPA1 and TRPV1, the receptor that is also activated by capsaicin, protons, and noxious heat.2,3 Both receptor channels are expressed in peptidergic nociceptive neurons, and activation, inducing calcium influx, can cause release of the proinflammatory neuropeptides calcitonin gene-related peptide (CGRP) and substance P from peripheral nerves.4,5 CGRP and substance P are the hallmark of “neurogenic inflammation,” including vasodilatation and plasma protein extravasation, respectively. Many recent studies report functional expression of TRPV16–9 and TRPA110–13 in sensory nerve endings of the airways, which can promote airway neurogenic inflammation and facilitate spastic reflexes.
However, there is no direct evidence that anesthetic gases actually reach the subepithelial nerves in the airways in sufficient concentration to activate TRP channels and release neuropeptides. From clinical observations, it is known that desflurane and isoflurane do act as irritants, whereas sevoflurane and halothane are considered nonirritant anesthetics.14 In the present study, we evaluated the anesthetic gases in an ex vivo animal model mimicking clinical exposure and allowing measurement of stimulated CGRP release as an index of tracheal nociceptor activation. The volatile anesthetics were gently blown onto the isolated superfused mouse trachea, and various mouse mutant strains were used to verify the involvement of both TRPA1 and TRPV1 in the evoked CGRP release.
METHODS AND STATISTICAL ANALYSIS
The experiments using animal donors were performed in accordance with the guidelines of the International Association for the Study of Pain. Adult C57BL6, TRPV1−/−, TRPA1−/−, and TRPA1/V1=/= double knockout mice were used. Breeding pairs of TRPV1 and TRPA1 knockout mice were obtained from Drs. John Davis15 and David Corey16 and continuously backcrossed to C57BL/6. Double knockout animals were generated in our animal facility by crossmating knockouts of both strains. The mice were housed in group cages in a temperature-controlled environment on a 12-hour light/dark cycle and were supplied with water and food ad libitum. Mice of either sex (body weight 15–25 g) were killed by exposure to increasing CO2 atmosphere (approved by the Animal Protection Authority, District Government Mittelfranken, Ansbach, Germany).
The trachea was excised together with the 2 main bronchi, hemisected along the dorsal midline, and mounted upside down for superfusion to mimic the natural direction of the mucus flow. A triangular aluminium block placed on a thermostatic plate (40°C providing at the tracheal surface 38°C) supported the preparation that was pinned in a Sylgard-lined groove in the falling slope (45°), which ended with a trough to collect the superfusate. Continuous superfusion (30 μL/min) by a thin layer of fluid was provided by a calibrated syringe pump (for microdialysis) linked to the tissue with a band of blotting paper. Synthetic interstitial fluid (SIF) was used for superfusion, containing (in mM) 107.8 NaCl, 3.5 KCl, 1.53 CaCl2, 0.69 MgSO4, 26.2 NaHCO3, 1.67 NaH2PO4, and 9.64 sodium gluconate. The superfusate was collected every 5 minutes and entered into the CGRP enzyme immunoassay (EIA) procedure. The trachea was stimulated for 5 minutes after 10 minutes superfusion with SIF, with a continuous stream of experimental gas mixture containing various concentrations (multiples of human minimum alveolar concentration [MAC]) of volatile anesthetics in oxygen (25%) and nitrogen (100%).
The proportional mixtures of oxygen, nitrogen, and volatile anesthetics were provided by a Dräger Julian® anesthetic machine (Dräger Medical, Lübeck, Germany), filled into natural latex balloons, and transferred to the experimental setup. The gas mixture was applied at room temperature to the isolated trachea via a tube and an inverted pipette tip at a 2-mm distance. To provide a constant gas flow (0.6 L/min) on the tracheal preparation, an aquarium pump was integrated between the balloon and the nozzle.
CGRP Enzyme Immunoassay
The CGRP content of the superfusate was measured using commercial EIA kits with a detection threshold of 5 pg/mL (Bertin Pharma, Montigny-le-Bretonneux, France). For this purpose, 100 μL of sample fluid was stored on ice and mixed immediately after the superfusion period with 25 μL of 5-fold--concentrated commercial CGRP-EIA buffer that contained a proprietary cocktail of peptidase inhibitors. The additional CGRP-EIA procedures were performed after the end of the experiment; the antibody reactions took place overnight. The EIA plates were determined photometrically using a microplate reader (Dynatech, Channel Islands, UK). All results are presented as measured by the EIA in pg CGRP/mL SIF. Reducing interindividual variability and day-to-day baseline variability, the data were referred to the second individual baseline value (before stimulation). This value was subtracted from all 4 (or 5) data points of a typical experiment so that only the absolute change in CGRP release (Δ pg/mL) is displayed in the figures.
Statistical comparisons were performed using Statistica 7 software (Statsoft, Tulsa, OK). All time series of experimental CGRP values were first analyzed for the effect of volatile anesthetic stimulation compared with baseline using the nonparametric Wilcoxon matched pairs signed-rank test to ensure that eventual increases in CGRP release were significant, that is, P < 0.05 (2-sided). The normalized (i.e., Δ) CGRP values were then entered into a 1-way repeated-measures analysis of variance followed by the Scheffé test, focusing on the peak values of stimulated CGRP release to compare the different gas treatments. P < 0.01(2-sided) was considered statistically significant. Data points represent means ± SEM of the given number (n) of identical experiments on tracheae of different animals; P values reported are numerically exact. The minimal sample size was a priori chosen to be n = 6 based on extensive experience with CGRP release experiments. In case small effects or differences had to be demonstrated or excluded, n = 7 or 8 was a priori assigned. In 2 cases in which no effect of the gas treatment was expected and factually seen, the series of experiments was canceled after n = 4 (Fig. 1).
Desflurane and Isoflurane Induce Concentration-Dependent CGRP Release from the Isolated Mouse Trachea
A soft constant stream of gas was blown for 5 minutes onto the superfused mouse trachea preparation; to control for possible mechanostimulatory effects, room air (22–23°C) was used, which did not alter the basal neurosecretion of CGRP (n = 4, data not shown). Exposure of the isolated mouse trachea to the pungent anesthetic desflurane in 1.5% concentration did not induce significant CGRP release, but 3%, 6%, and 12% caused significant increases over baseline (P = 0.028, 0.018, and 0.028; Wilcoxon matched pairs signed-rank test). Desflurane 6%, corresponding to 1 MAC, already induced the maximal tracheal response, greater than at 3% (0.5 MAC; Fig. 2). Desfluran 12% (2 MAC) seemed less effective than 6% in releasing CGRP (P = 0.41), suggesting an inversely U-shaped or saturating concentration–response relationship. The pungent anesthetic isoflurane at a concentration of 0.6% did not induce significant tracheal CGRP release (P = 0.5), but isoflurane 1.25 % and 2.5% caused significant increases (P = 0.028 both; Wilcoxon test). Isoflurane 1.25%, which is 1 MAC for this gas, showed the greatest tracheal CGRP release, 2.5% (2 MAC) again similar to 12% desflurane caused a smaller effect (Fig. 1). The irritant effect of 1 MAC isoflurane was just half the effect of 1 MAC desflurane (compare Figs. 1 and 2). This difference between desflurane and isoflurane did not reach statistical significance (P = 0.085) but may be considered indicative for less irritancy of isoflurane at equal anesthetic concentration. Exposure of the isolated mouse trachea to the nonpungent volatile anesthetic sevoflurane in a concentration of 2% (1 MAC) caused no significant release of CGRP (Fig. 1, black trace).
Oxygen Fraction Has No Influence on Desflurane-Induced CGRP Release
Tracheal CGRP release stimulated with a gas mixture containing 6% desflurane, 25% oxygen, and 69% nitrogen did not significantly (P = 0.49) differ from the response achieved with a gas mixture containing 6% desflurane and 94% oxygen (Fig. 3). The response magnitude was far from being maximal (“ceiling”) because for example, strong nicotine stimulation of the trachea can release 120 pg/mL CGRP; also, additive effects from various irritants are to be measured in this model.17 The oxygen experiment was inspired by a recent proposal, based on cellular models, that TRPA1 could serve as a sensor for hyperoxia, explaining the well-known irritancy of pure oxygen and part of its cytotoxicity.18 In our ex vivo trachea model, this theory found no support.
Desflurane-Induced CGRP Release Is TRPA1 and TRPV1 Dependent
For the experiments to test involvement of the TRP channels in tracheal CGRP release, the pungent desflurane was chosen in the maximal effective concentration of 6% (1 MAC). TRPV1−/− mice showed a substantially reduced response to desflurane compared with wild-type mice. In TRPA1−/− and TRPA1/TRPV1=/= double knockout mice, the CGRP response was completely absent (Fig. 4). Thus, the desflurane irritancy in the mouse trachea depends on both TRP receptor channels, although the contributions of each are not additive. TRPA1 seems to be the predominant irritant sensor for pungent anesthetic gases and inducer of neurogenic inflammation.
Our results from measuring (basal and) stimulated CGRP release from the isolated superfused mouse trachea support the clinical observation that desflurane and isoflurane, but not sevoflurane, irritate nociceptive tracheobronchial nerve endings. At an equally anesthetic concentration (1 MAC), isoflurane appeared less irritating than desflurane, which is also in accord with clinical experience.14 A high oxygen concentration (94%) did not augment the irritancy of desflurane. Importantly, desflurane exerted its effects through both irritant receptor channels TRPA1 and TRPV1, which probably also applies to the structurally related isoflurane.
Isoflurane has been shown to evoke a TRPA1-dependent, neuropeptide-mediated constriction of isolated guinea pig bronchi and to activate recombinant rat TRPA1.2,19 However, heterologously expressed rTRPV1 was not activated by even 0.9 mM (2.9 MAC) isoflurane, whereas in our hands, 1 MAC of isoflurane was a supramaximal stimulus to induce tracheal CGRP release. The closely related and more irritating desflurane also showed a maximal effect at 1 MAC, and this was clearly reduced (by ~75%) in TRPV1-deficient mice. This suggests a discrepancy (with Matta et al.,2) although we are comparing isoflurane with desflurane, which may be allowed, given their similar structure–activity relationship. Yet, the discrepancy may be explained by the fact that calcium imaging and patch clamping of transfected cells were done at room temperature, whereas our ex vivo experiments are run at 38°C. TRPV1, a heat-activated ion channel, is markedly sensitized by increasing temperature and inhibited by decreasing it, whereas TRPA1 is desensitized by warming.20–23 Such a sensitization of TRPV1 can also be achieved (at room temperature) by the inflammatory mediator bradykinin and other compounds activating protein kinase C, and it applies not only to classical TRPV1 stimuli such as capsaicin, protons, and heat but also includes desflurane and isoflurane, which are able to activate the sensitized TRPV1.24 Thus, at (mouse) body temperature and under inflammatory conditions, the balance between the 2 TRP irritant receptors is shifted toward TRPV1.
The irritant effect of desflurane on the trachea was abolished in TRPA1 null mutants (and double knockouts) but only reduced (to 25%) in TRPV1-deficient mice. The TRPA1-expressing sensory neurons are generally considered a subpopulation of TRPV1-positive neurons, based on immunocytochemical findings.25 This has largely been confirmed with single-cell reverse transcription polymerase chain reaction on nodose-jugular ganglion neurons projecting to the lungs; however, in 1 of the 10 TRPV1-negative cells, TRPA1 mRNA was clearly identified.5 This is compatible with several of our recent calcium-imaging studies on sensory neurons, in particular, with 1 on nodose-jugular ganglia, in which a consistently small subpopulation of cells responded to allyl isothiocyanate or another TRPA1 agonist but, surprisingly, not to the TRPV1 agonist capsaicin.3,17,26–28 These may be the neurons that provide the small desflurane response retained in TRPV1 null mutants. However, 12 of 12 cells in the single-cell polymerase chain reaction study coexpressed TRPV1 and TRPA1, which raises the question: Why would all desflurane-induced CGRP release be abolished in TRPA1 knockouts, but TRPV1 is retained as an effective transducer? Only half of the TRPV1-expressing nerves in the (guinea pig) trachea are immunopositive for neuropeptides and, thus, can contribute to stimulated CGRP release.6 However, a more complete answer to the above question would require a deeper understanding of the multiple and mutual interactions between TRPA1 and TRPV1 in coexpressing neurons than are presently available.29 Phenomenologically, it appears from our data that TRPV1 is disabled to transduce the irritant desflurane effect in the absence of TRPA1. In contrast, other selective TRPV1 agonists such as capsaicin and the IV irritant anesthetic propofol retain their efficacy in TRPA1 null mutants.3,16
Both isoflurane and desflurane showed a ceiling effect at 1 MAC, which may mean that the necessarily higher anesthetic gas concentrations of up to 3 MAC do not augment the adverse irritant effects. In this respect, isoflurane appeared less irritating than desflurane, whereas sevoflurane at 1 MAC did not cause measurable tracheal CGRP release; at equal 1.8 MAC, both sevoflurane and desflurane suppress the mechanically evoked tracheal cough response.30 The latter action of volatile and other general anesthetics is attributed to their central nervous system–depressant effects, including activation of the ionotropic γ-aminobutyric acid type A (GABAA) receptors, in particular, by propofol.3,31,32 In primary afferent neurons, GABAA receptors are functionally expressed in the somata and in the presynaptic terminals of the spinal dorsal horn and brain stem, in which activation of the channel causes chloride outflow, depolarization, inactivation of voltage-gated sodium channels, and, thus, finally presynaptic inhibition. Depolarization of peripheral nerves, for example, by KCl, releases CGRP; however, neither GABA nor the selective GABAA agonist muscimol did so in wild-type mice, nor did the irritant propofol release CGRP from the nerves of TRPA1-TRPV1 double knockouts.3 Thus, GABAA 1 of the targets of volatile anesthetics does not seem to be efficiently expressed in the periphery.
Although the previous cellular and molecular study on irritant volatile anesthetics first overlooked the TRPV1 contribution, a clinically important observation was made measuring the degree of experimental inflammation, depending on the anesthetic used. Isoflurane was more than twice as effective as sevoflurane in augmenting the neurogenic inflammatory effect of topical allyl isothiocyanate on the mouse ear.2 This indicates a systemic, sensitizing, and proinflammatory effect of the irritant anesthetics with negative impact on postoperative periods. Cooling the surgical region is an approved remedy, blunting TRPV1, but tranquilizing TRPA1 pharmacological aid will be required.
Name: Tatjana I. Kichko, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Tatjana I. Kichko 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: Florian Niedermirtl, MD.
Contribution: This author helped design the study and conduct the study.
Attestation: Florian Niedermirtl has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Andreas Leffler, MD, PhD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Andreas Leffler has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Peter W. Reeh, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Peter W. Reeh 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.
This manuscript was handled by: Jianren Mao, MD, PhD.
We thank Susanne Haux-Oertel, Annette Kuhn, and Jana Schramm for excellent technical assistance, genotyping, and breeding the knockout mice.
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