The precision-cut lung slice (PCLS) technique is widely used to examine airway responses in different species. We developed a method to study nerve-dependent bronchoconstriction by the application of electric field stimulation (EFS) to PCLS. PCLS prepared from Wistar rats were placed between two platinum electrodes to apply serial rectangular impulses (5–100 Hz), and bronchoconstriction was studied by videomicroscopy. The extent of airway contractions increased with higher frequencies. Stable repeated airway contractions were obtained at a frequency of 50 Hz, a width of 1 ms, and an output of 200 mA for 2.5 s each minute. Larger airways showed stronger responses. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine (10 μM) and reversed by the muscarinic antagonist atropine (10 μM), whereas the thromboxane protanoid receptor antagonist SQ29548 (10 μM) had no effect. Magnesium ions (10 mM) antagonized airway contractions induced by EFS, but not by methacholine, indicating that nerve endings remain intact in PCLS. Our data further show that the electrically evoked airway contractions in PCLS are mediated by cholinergic nerves, independent of thromboxane and more prominent in larger airways. Taken together these findings show that nerve endings remain intact in PCLS, and they suggest that the present method is useful to study neurogenic responses in airways of different size.
- small airways
- muscarinic receptor
- innervation of the lung
the mammalian lung is innervated by sympathetic adrenergic, parasympathetic cholinergic, and nonadrenergic, noncholinergic nerves (NANC) (10). Irritation of denudated nerves in inflamed lungs is likely to contribute to the airway hyperresponsiveness typical for asthma, with reflex smooth muscle contraction, mucus secretion, and edema formation (38). The parasympathetic nervous system is the dominant bronchoconstrictor mechanism in all animals (6). Acetylcholine is released from nerve terminals and binds to muscarinic (M) receptors at different sites throughout the lungs. The M1 receptor is localized on parasympathetic ganglia and is thought to primarily facilitate cholinergic neurotransmission by nicotinic acetylcholine receptors. M2 receptors on the presynaptic nerve terminals control acetylcholine release in a feedback manner, limiting further acetylcholine liberation. Moreover, M2 and M3 receptors are found on the airway smooth muscle (ASM) cells. M3 receptor activation leads to ASM contraction, a process facilitated by M2 receptors by counteracting cAMP-mediated relaxant pathways (7). In addition, inflammatory mediators, e.g., leukotrienes or thromboxane, have been reported to enhance acetylcholine release by activating afferent sensory nerve fibers and by directly facilitating ganglionic neurotransmission and acetylcholine release from vagal nerve terminals (2, 3, 7, 29, 37, 39).
Application of an electric field across isolated airway preparations results in activation of intrinsic nerves, the release of neurotransmitters, and response of the ASM to these neurotransmitters (15). Electric field stimulation (EFS) has been studied in three airway preparations; i.e., tracheal strips, bronchial strips, and parenchymal strips (15), which were mounted in organ baths and faced by parallel platinum electrodes. These models have been extremely useful for understanding the neural control of airway tone in different species; e.g., mouse, rat, guinea pig, horse, or humans among others (4, 5, 14, 17, 19, 22, 32, 40, 41), and remain important (8, 32).
The organ bath method also has some disadvantages. First, it can be applied only to relatively large airways and preparations from the lower airways of smaller mammals are difficult to obtain; e.g., EFS-based studies in rats have been possible only until airway generation 8 (15). However, innervation is unevenly distributed along the tracheobronchial tree. For instance, in the guinea pig trachea, cholinergic nerves predominate over tachykinergic nerves, whereas the latter predominate in the bronchus (4). Therefore, methods to examine smaller sized airways in different species are needed (31). Second, organ bath methods are often performed under either isometric or isotonic conditions and require a muscle preload. This creates potential problems: the preload is artificial and may influence the result (15) or even damage the smooth muscle (15). Furthermore, contractions are not auxotonic as in vivo, where the ASM shortens against an increasing load imposed by the attachments of the surrounding lung parenchyma, parallel elastic elements (i.e., extracellular matrix), and mucosal folding (11, 15, 23). More recently, some of these problems have been solved by the development of servocontrolled force-length transducers that adjust airway preparations to match a muscle length that was determined before the specimen was prepared (8, 9). Third, smaller airways require increasingly sensitive equipment. Spiral tissue preparations, used to increase contractions, are hampered by misalignment of the smooth muscle from the longitudinal axis and force generation is lost to the coil (15). Fourth, organ bath experiments require a relatively large buffer volume, generally more than 5 ml, which is a disadvantage in case of expensive substances. Finally, in whole tissue preparations (i.e., parenchymal strips), contractions from airways or vessels cannot be distinguished (15).
All these problems are addressed in precision-cut lung slices (PCLS): small airways are accessible, airway contractions are more auxotonic, preload is given by the tissue, force measurements are not required, the required buffer volumes are small, and airways and vessels can be easily distinguished (11, 25, 26). Given the importance of small airways for asthma and chronic obstructive pulmonary disease (COPD) (36, 42, 43), the possibility to study different airway generations in PCLS is also of clinical relevance. In the present study, EFS was applied to PCLS for the first time. PCLS from rats responded to EFS indicating that nerve endings remained intact in this preparation. The electrically triggered airway contractions were of neural origin and mainly cholinergic. This novel method allowed us to study the functional consequences of nerve activation in airways of different size and revealed a significant correlation between airway size and neurally induced airway contraction. Moreover, we showed that thromboxane prostanoid (TP) receptors exert no effect on the muscarinic airway contractions induced by EFS or methacholine.
MATERIALS AND METHODS
Lungs were prepared from female Wistar rats weighing 200–300 g. Rats were obtained either from Harlan Winkelmann (Borchen, Germany) or Janvier (Le Genest, St. Isle, France) and were kept under controlled conditions in the animal house. Animal experiments were approved by the local ethic committee (Reference number 8.87–51.05.20.09.245).
Acetylcholine, atropine, methacholine, neostigmine, magnesium sulfate (MgSO4), and standard laboratory chemicals were purchased from Sigma (Steinheim, Germany). SQ29548 and U46619 were provided by Biomol (Hamburg, Germany). Substances for cell culture were obtained from PAA laboratories (Cölbe, Germany).
Preparation of PCLS.
PCLS were prepared as described (43) and used 1 day after preparation. Briefly, the lungs were filled with low-melting agarose after tracheotomy and cooled on ice. The hardened lung was removed en bloc from the animal and lobes were separated. Tissue cores with a penetrating airway in its center were prepared from the left and right inferior lobe by a rotating sharpened metal tube (diameter 10 mm). Tissue slices were cut from the cores perpendicular to the airway by the aid of a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL). Only slices free of agarose, with beating cilia and an intact smooth muscle layer in a relaxed state, were used (Fig. 1C).
Airway area was monitored by videomicroscopy (SensiCam 365KL, Visitron Systems, Munich, Germany) at a frame rate of 0.36 s−1 in experiments with EFS and 0.2 s−1 in all other experiments. Camera control and image analysis were achieved by Optimas 6.5 software (Optimas, Bothell, WA). Airway area was defined as 100% area size before the first stimulation (Fig. 1D).
Electrical field stimulation.
EFS of PCLS were carried out in cavities of standard 12-well plates in a reaction volume of 1 ml standard minimal essential medium [2 mM CaCl2, 1 mM MgSO4, 5 mM KCl, 116 mM NaCl, 1 mM NaH2PO4, 17 mM glucose, 26 mM NaHCO3, 25 mM HEPES, 1 mM sodium pyruvate, 2 mM glutamine, amino acids (PAA Laboratories, M11-002, 1:50) and vitamins (PAA Laboratories, N11-002, 1:100)]. The PCLS (10 mm in diameter) were placed between two platinum electrodes of 12 mm distance. In the established PCLS model, PCLS are always pinned down by a nylon thread fixed to a platinum wire (25). However, this approach was not possible here as the platinum wire alters the electric field, therefore, we used a Teflon ring instead (9 mm outer diameter × 6 mm inner diameter × 4 mm height) (Fig. 1A). The electric field was applied by a HSE Stimulator II (Hugo Sachs Electronics, March Hugstetten, Germany). Our standard provocation protocol comprises a stimulation train lasting 3.3 min.
In preliminary experiments the following setting were identified as useful: train rhythm (TR) = 60 s, train width (TW) = 2.5 s, frequency (F) = 50 Hz, pulse duration (B) = 1 ms, current (A) = 200 mA (Fig. 1B). To systematically examine the best settings for frequency, pulse duration, current, and train width, PCLS were stimulated by modulation of one parameter, while the others were kept constant at the values noted above. Frequency, pulse duration, current, and train width were examined at 5–100 Hz, 0.1–5 ms, 1–200 mA, and 0.5–20 s, respectively. Recovery between the repetitive stimulations lasted until the airway had relaxed to its initial state (3–5 min).
Cholinergic contribution to airway contraction in EFS.
Each PCLS was exposed to three stimulation trains. The first stimulation train was carried out in the absence of any additive, the effect of acetylcholine esterase inhibition (10 μM neostigmine) was examined in the second train, and the role of acetylcholine receptors (10 μM atropine) was studied in the third train. All drugs were added 15 min before stimulation.
Neural contribution to airway contraction in EFS.
PCLS were repeatedly stimulated by EFS. The first stimulation train occurred at 10 μM neostigmine. Half an hour later the second stimulation occurred either in the absence or presence of 10 mM MgSO4 to block neuronal calcium entry. MgSO4 was present 30 min before stimulation. After the last stimulation, 10−4 M acetylcholine was added to examine airway contractility in the presence of 10 mM MgSO4, and responses were monitored at a frame rate of 0.2 s−1 for 5 min.
Magnesium in exogenously evoked ASM contraction.
A dose-response curve was conducted with methacholine (10−8-10−4 M) in the presence or absence of 10 mM MgSO4. PCLS were preincubated with MgSO4 30 min before addition of the first methacholine concentration. Methacholine was cumulatively added for 5 min each.
Examination of large and small airways in EFS.
PCLS containing differently sized airways ranging from 3.8 × 103 to 1.2 × 106 μm2 (0.07–1.2 mm in diameter) were stimulated by EFS. The first stimulation train was conducted without additive, and the second train was carried out in the presence of 10 μM neostigmine given 15 min in advance.
Effect of TP receptor antagonist SQ29548 in EFS.
PCLS were repeatedly stimulated by EFS. The first stimulation train occurred at 10 μM neostigmine. Half an hour later the second stimulation occurred either in the absence or presence of 10 μM SQ29548 to block TP receptors. SQ29548 was applied 15 min before stimulation. After the second stimulation, 10−5 M U46619 was added to prove receptor blocking by SQ29548 and to verify a priming of acetylcholine release by U46619 in a following third EFS train. Airway responses during U46619 application were monitored at a frame rate of 0.1 s−1 for 15 min.
Atropine in U46619 evoked ASM contraction.
A dose-response curve was conducted with U46619 (10−8-10−4 M) in the presence or absence of 10 μM atropine. Preincubation of atropine occurred for 15 min before addition of the first U46619 concentration. U46619 was cumulatively added every 5 min.
Data are expressed as means ± SD. Nonlinear regression, Spearman correlation, unpaired t-test, t-test with Welch's correction, Mann Whitney test, or Tukey's multiple comparison tests were performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA).
PCLS were placed between two electrodes, and reduction in airway lumen area became apparent after electric stimulation (Fig. 2A). The bronchoconstriction was reversible as long as the Teflon ring was used (Fig. 2A). Contractions increased in a frequency-dependent manner up to 50 Hz, (Fig. 2B) with a half-maximal response (EF50) at 16.7 ± 4.9 Hz. Bronchoconstriction became stronger with pulse duration, current, and train width. A plateau response for pulse duration was reached at pulses ≥ 1 ms (Fig. 2C). At 200 mA, the maximal current output of the stimulator, airways contracted about 35% (Fig. 2D). Maximal ASM contraction occurred if train width lasted 10 s or longer (Fig. 2E). However, the recovery phase to reach the initial airway area lasted longer at TW = 10 s, and contractions persisted in presence of 10 mM magnesium (data not shown), suggesting unspecific nonneural stimulation at this train width. We therefore decided to carry out the following experiments with the Teflon ring in place, at F = 50 Hz, B = 1 ms pulse duration, A = 200 mA, and TW = 2.5 s. These conditions resulted in reproducible airway contractions (Fig. 3).
To examine the role of cholinergic nerves in the EFS-induced bronchoconstriction, PCLS were stimulated in the presence of neostigmine to enhance acetylcholine concentrations in the synaptic cleft and atropine to block postsynaptic acetylcholine receptors. In the presence of neostigmine (10 μM), EFS contracted airways to 49.2 ± 11.0% of the initial airway area compared with 84.1 ± 5.3% without neostigmine (Fig. 3). With neostigmine still present, atropine nearly completely abolished the EFS-induced airway contractions (Fig. 3; see supplementary video 1 online). Similar observations were made for pulse durations ranging from 0.5 to 2 ms (data not shown). These findings indicate that airway contractions in rat PCLS mainly depend on muscarinic receptors.
To verify that the EFS-evoked airway contractions were due to neural stimulation, we examined the effects of magnesium concentrations high enough to block voltage-gated calcium entry in neuronal cells (12, 16). MgSO4 (10 mM) totally abolished the EFS-induced contractions in the presence of 10 μM neostigmine (Fig. 4, A–C; see supplementary video 2 online). In contrast, airway contractions by exogenously added acetylcholine were not affected by MgSO4 (Fig. 4, A–C), and 10 mM MgSO4 did not alter the concentration-response curve of methacholine-induced bronchoconstriction (Fig. 4D).
Since small airways respond stronger to methacholine than larger ones (25), we analyzed the response of small and large airways to EFS (Fig. 5). EFS were studied in PCLS with airway sizes ranging from 3.8 × 103 to 1.2 × 106 μm2 in the presence or absence of neostigmine. Our findings show that larger airways were more responsive than smaller ones (Fig. 5). Additionally, the amplification of airway contraction by neostigmine was more pronounced in larger airways.
An interaction between TP receptor signaling and parasympathetic cholinergic acetylcholine release has been shown for the dog, guinea pig, and mouse (2, 3, 37, 39), whereas this mechanism has not yet been characterized in rat lungs. We therefore examined the influence of the TP receptor antagonist SQ29548 in EFS of rat PCLS. No difference was found for electrically evoked cholinergic airway responses in the presence and absence of a TP receptor antagonist (Fig. 6). A statistical power calculation based on these results (Fig. 6C) indicated that at least 32 experiments were needed to possibly reach significance, indicating that any role of TP receptors would be rather small. The addition of U46619 demonstrated that SQ29548 was capable of blocking TP receptors in our system (Fig. 6D). Moreover, a subsequent EFS train in the presence of U46619 did not increase the response (Fig. 7). Vice versa, the influence of TP receptor agonist U46619 and its possible interactions with muscarinic receptors was studied by the use of atropine. The airway responses for cumulative concentration-response curves with U46619 were the same in the presence and absence of atropine (Fig. 8).
Viable PCLS have been established as a useful tool to study airway tone, pulmonary vascular responses, ciliary beating, as well as immunological and toxicological properties of the lungs (11, 18, 25, 26, 42, 43). Here we show that neural stimulation of PCLS is feasible and that the EFS-induced bronchoconstriction is consistent, reproducible, sensitive to atropine, and enhanced by an acetylcholine esterase inhibitor in rats. The effect of the EFS was strongest in the larger airways and appeared to be mediated by synaptic release of acetylcholine. This model will be useful to further study mechanisms of neural airway control and cholinergic airway hyperreactivity in rats and other species.
General EFS setup in PCLS.
The extent of the EFS-induced contractions of 15% in large airways roughly corresponds to concentrations elicited by 330 nM of the metabolically stable acetylcholine derivative methacholine [Martin et al. (25), Fig. 8B], suggesting that this may be the effective acetylcholine-concentration in the synaptic cleft following presynaptic discharge. These findings propose that neurally induced bronchoconstriction can regulate airway tone but is unlikely to lead to complete airway closure as shown, for instance, for allergen provocation (43). When comparing the present findings with other studies, it should be noted that we did not normalize our data to contractions induced by KCl, a procedure that may seemingly increase the magnitude of the effects. Normalization to receptor agonists (e.g., methacholine) was omitted throughout the study as we have previously shown stronger responsiveness of small airways to methacholine and other stimuli (25, 26, 42, 43). In such a situation, normalization would distort the results. The EFS-induced contraction by ∼15% was strongly potentiated by inhibition of the acetylcholine esterase (Fig. 3). These findings indicate that in PCLS cholinergic synapses remain intact, as they respond to electrical currents, possess active acetylcholine esterase (see experiments with neostigmine) and acetylcholine-receptors (see experiments with atropine), and recycle acetylcholine in a normal fashion (otherwise repetitive stimulations were not possible).
EFS was applied to stimulate neuronal cells, but depending on the frequency and duration of the electric stimulus, direct contraction of smooth muscle cells may also occur (13). To exclude such an unspecific direct stimulation of voltage-gated channels on the airway smooth muscle, we examined the effect of magnesium. At high concentrations, magnesium prevents voltage-induced calcium entry and thereby the release of neurotransmitters from nerve terminals (12, 16). Since magnesium completely prevented the bronchoconstriction induced by EFS but not by methacholine, we conclude that in our setup the EFS acts only on nerve endings and not on airway smooth muscle (Fig. 4).
Role of frequency, pulse duration, current density, and train width in EFS.
For organ bath experiments, Wang and colleagues (40) systematically studied the influence of pulse duration (0.5–3 ms), muscle preload (2–20 g), voltage (5–20 V), and frequency (0.5–16 Hz) on the release of acetylcholine from equine airway cholinergic nerves, to provide guidelines for selecting EFS parameters in future studies. Because they observed an increasing acetylcholine release from 2.1 to 18.6 pmol·gtissue-wet-wt−1·min−1 with increasing frequencies (2–16 Hz), we analyzed this frequency range while the stimulation period was kept constant. This resulted in a sigmoidal frequency-response curve for the airway area change (Fig. 2B) plateauing at F = 50 Hz, somewhat above the frequency observed by Wang and colleagues (40) (>8 Hz). The most likely explanation for these differences is interspecies variability, as PCLS from other species (e.g., sheep) responded already at lower frequencies (data not shown), and our EF50 of 16.7 ± 4.9 Hz is almost identical to the 19.3 ± 4.3 Hz reported by Gonzalez and Santana (17) on isometric tension studies of rat tracheal smooth muscle. In addition they also found a plateau phase above 40 Hz (17), whereas the EF50 of 4.4 Hz in Wang's and colleagues (40) study of horse airways was markedly lower. To put these figures into proportion, the maximal frequency that allows motoneurons to depolarize during repetitive stimulation is 500–1,000 Hz (21). It should also be noted that Mitchell and colleagues (30) showed relatively high frequencies of action potentials (peak frequency of 10–13 Hz) for feline tracheal parasympathetic ganglion cells during normal breathing. Also other factors such as electrode geometry and the ionic strength of our cell culture medium have to be taken into account, as the inner electric field strength depends on the conductivity of the medium and on the current density, which in turn depends on a shape factor, the frequency, and the external electric field strength. Taken together, the frequencies that were applied in rat PCLS appear reasonable.
With respect to pulse duration we found, similar to Wang and colleagues (40), that durations B ≥ 1 ms were needed to reach a plateau response. In all our experiments (except Fig. 2D) we used a current density of A = 200 mA, which was the only current that gave a reproducible contraction; since 200 mA is the maximum current output of the HSE-stimulator II, higher currencies could not be studied. Another important factor influencing ASM contraction is train width: here maximal contraction was achieved at TW ≥ 10 s, similar to Bosse and colleagues (8, 9), who used a maximum airway contraction at TW = 9 s when examining the influence of increased tone (by acetylcholine) on the mechanical properties of ASM. However, at TW = 10 s, airway contractions took longer to reverse and, more importantly, could not be blocked completely by magnesium, indicating that the electrical stimulation was unspecific at this train width and activated both nerve endings and airway smooth muscle. Therefore, we decided to use TW = 2.5 s to avoid direct activation of airway smooth muscle, but a TW = 5 s would also seem appropriate. Since we used F = 50 Hz, a TW = 2.5 s implies a series of 125 single pulses that were separated by a recovery phase of about 1 min (exactly 57.5 s). The duration of this phase allowed recovery under normal conditions (Fig. 2A; Fig. 3, top) but was too short when neostigmine was present. However, as the maximum contraction was always reproducible, we conclude that the lack of complete recovery in Figs. 3, 4, and 6 is not a major limitation. It is important though to note that the different stimulation trains were separated by at least 15 min to assure full recovery of the ASM to its physiological ground state before the next experimental setting was examined. In addition this period was used to equilibrate the slices with the pharmacological agents to be studied next.
Cholinergic neuronal response in PCLS.
Acetylcholine release from parasympathetic nerve terminals regulates the airway tone in various species including humans (6, 10, 17). The enhanced airway contraction after addition of neostigmine, an acetylcholine esterase inhibitor (Fig. 3), indicates locally increased concentrations of acetylcholine at the neuroeffector junction (24, 27). Atropine, an unspecific inhibitor of muscarinic receptors, almost completely inhibited the neuronal cholinergic contraction in PCLS, even in the presence of neostigmine (Fig. 3). Atropine most probably prevents ASM contraction by blockade of the M3 (and M2) receptors on the postsynaptic membrane. Thereby the M3 receptors cannot initiate the contraction through the inositol trisphophate pathway (6, 7, 28, 33). The small residual bronchoconstriction in the presence of 10 μM atropine may be explained by the blockade of presynaptic muscarinic receptors by atropine, preventing the autoinhibitory effect of acetylcholine release (6, 7), and/or the release of substance P that might potentiate the release of acetylcholine from cholinergic nerves (6) and trigger the release of bronchoconstrictive tachykinins from excitatory NANC (14).
Reactivity of different airway generations.
PCLS have previously been used to study airway responses along the bronchial tree. Smaller airways are more responsive to methacholine, serotonin, the TP receptor agonist U46619, and allergens in lungs of different species (26, 42, 43). Endothelin-1 contracted airways independent of their size (26), indicating that smaller airways are not more sensitive in principle. Here we show that smaller airways are less sensitive to EFS (Fig. 5). This observation can be explained most likely by the decreasing innervation of the bronchial tree toward the peripheral regions (6). Thus parasympathetic bronchoconstriction in the airways appears to be balanced by inversely arranged innervation and acetylcholine responsiveness: rich innervations with low acetylcholine responsiveness in large airways opposed to more sparse innervations with high sensitivity in smaller airways.
Role of TP receptor in neuronal activation.
Subthreshold doses of TP receptor agonists (e.g., U46619); i.e., doses that do not evoke airway contraction via the TP receptor themselves, have been shown to increase parasympathetic cholinergic bronchoconstriction in mice (as measured by RL) (3), guinea pig (RL) (39), and dogs (isometric tension) (2, 37). Interestingly, in the study of Takata and colleagues (37) the effect was only found for bronchial smooth muscle, whereas tracheal smooth muscle was unaffected suggesting regional differences in airway responsiveness, with smaller airways being more susceptible to thromboxane. Additionally, Saroea and colleagues (35) suggested U46619-induced bronchoconstriction via acetylcholine release in asthmatic subjects. Moreover, rat tracheal prostanoid synthesis can be stimulated by activation of muscarinic receptor-linked Ca2+ mobilization (20). These observations suggested a possible influence of endogenously released thromboxane on EFS in our model. However, the TP-receptor antagonist SQ29548 had no effect (Fig. 6), indicating that EFS-induced airway contractions in the rat are not influenced by thromboxane. This is confirmed by the observation that even a precontracting concentration of U46619 did not alter the response to EFS (Fig. 7). These observations are in line with other in vitro studies. For instance, Aizawa and Hirose (1) measured airway contraction of canine tracheal strips in response to acetylcholine or EFS either in presence or absence of PGF2α or stable thromboxane A2 and found no difference. They therefore suggested that the interaction of thromboxane and cholinergic pathways depends on stimulation of vagal sensory endings and activation of the reflex pathway in vivo. This conclusion is supported by studies from Underwood and colleagues (39), who discriminated strictly between in vitro and in vivo responses to PGD2 and 9α,11β-PGF2. In vivo, atropine antagonized prostaglandin increased airway resistance similar to the TP receptor antagonist SK&F 88046, whereas in vitro (tracheal strip) only TP receptor antagonists were effective. Thus our findings suggest that a complete reflex arc is not present in PCLS.
An interaction of TP- and muscarinic receptor pathways may not be limited to prejunctional nerves but may also occur at the ASM itself. We therefore conducted cumulative dose-response curves with U46619 in presence or absence of atropine and found no difference (Fig. 8). This finding is in accordance with in vitro contraction of tracheal strips from guinea pigs, where comparable dose-response curves for U44069, PGD2, and 9α,11β-PGF2 in the presence or absence of atropine were obtained (39). In contrast, Allen and colleagues (3) observed a notable reduction in U46619 (50–500 nM)-evoked murine tracheal ring constriction at 1 μM atropine. They explain their findings by an increased sensitivity of the ASM M3 receptor to its cognate physiological ligand acetylcholine after stimulation of the ASM TP receptor by thromboxane. However, their model presupposes the existence of an ASM basal tone and constitutive release of acetylcholine in the unprovoked airways. Therefore, their data are not conflicting to our results, because we assume that there is no or negligible release of acetylcholine in our system, since neostigmine had no effect in unstimulated PCLS (Fig. 3). Nonetheless, possible species differences can easily be studied with the help of PCLS (11, 25, 34, 42, 43).
Comparison to other in vitro models.
Previously, EFS has been almost exclusively studied in the organ bath using tracheae, bronchi, and parenchymal strips. In comparison, PCLS have both advantages and disadvantages.
First, in the organ bath, studies are limited to the trachea or upper bronchi or to the very distal part of the lungs as in parenchymal strips. PCLS from rat lungs allow to study nearly all generations along the bronchial tree. Though PCLS are very thin and therefore the percentage of neurons and especially ganglia will be lower than in tracheal and bronchial preparations. Second, airway contractions in the organ bath are either isotonic or isometric, whereas it is more auxotonic (like in vivo) in PCLS. The airways remain embedded in the parenchyma that exhibits tethering forces on the airways increasing with contraction. These tethering forces are enhanced by using a Teflon ring that weighs on the outer parts of the slice, so that the tethering forces increase during airway contractions. In the absence of the Teflon ring the whole slice contracted and no relaxation occurred, implying the lack of sufficient tethering forces (Fig. 2A). So presumably, the Teflon ring acts like the attachment of the lung tissue to larger vessels and airways. Third, our PCLS setup requires only small buffer volumes of 1 ml, which can be further decreased to 0.4 ml. Fourth, PCLS allows one to analyze airway responses throughout the bronchial tree, which allows one to analyze airways down to a diameter of 50 μm (25, 42, 43). Thus the present method will help to further elucidate the role and the mechanisms of small airway innervation. In contrast to classic parenchymal preparations for the study of peripheral lungs, in which force generation from different sources (ASM, vasculature) cannot be distinguished, our model can specifically attribute contractions to airways or vessels along the entire tracheobronchial tree. And finally, the technique of PCLS is easily transferred to other species, including humans.
In conclusion, the present study shows that PCLS respond to EFS, indicating that terminal nerves remain intact in this preparation. In rats EFS-induced airway contractions are mediated by cholinergic nerve stimulation and airway innervations decrease with airway size. EFS of PCLS may serve as a model to study neurally mediated responses in large and small airways.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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