Animals and human material/Ethics statement

Harvesting of lungs from laboratory animals was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (LANUV, approval-ID: 8.87–51.05.20.10.245). Balb/c mice (21–23 g) were obtained from Harlan Winkelmann (Borchen, Germany), Wistar rats (200–300 g) from Janvier (Le Genest St. Isle, France) and Dunkin Hartley guinea pigs (340–550 g) from Charles River (Sulzfeld, Germany). Lungs from adult marmoset monkeys (Callithrix jacchus) were obtained from the German Primate Center (Göttingen, Germany). Sheep lungs were taken from adult Texel sheep kept at the Maastricht University Medical Center (Maastricht, Netherlands). Care and housing conditions of the animals complied with the regulations of the European Parliament and the Council Directive on the protection of animals used for scientific purposes (2010/63/EU) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Human PCLS were prepared from patients undergoing lobectomy due to cancer. After pathological inspection, cancer free tissue from tumor-far parts of the lobes was used. The experiments were approved by the Ethik-Kommission an der medizinischen Fakultät der Rheinisch-Westfälischen Technischen Hochschule Aachen (RWTH Aachen) and all patients gave written consent.

Chemicals

Atropine, capsaicin, magnesium sulphate, methacholine, neostigmine, Ruthenium red, SKF96365 (1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole) and standard laboratory chemicals were purchased from Sigma (Steinheim, Germany). Substances for cell culture were obtained from PAA laboratories (Cölbe, Germany).

PCLS

PCLS were prepared as previously described [22] and used one day after preparation. Briefly, whole lungs (mouse, rat, guinea pig, marmoset) or lung lobes (sheep, human) were filled via the trachea or main bronchus, respectively, with 1.5% (w/v) low-melting agarose and cooled on ice to harden. Tissue cores (10 mm in diameter) with a penetrating airway in its center were prepared by a rotating sharpened metal tube and PCLS were cut perpendicular to the airway by means of a Krumdieck tissue slicer (Alabama Research and Development, Munford, Al, USA) filled up with slicing 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). As the lungs from mice were too small for tissue coring, the left and right lung were separately embedded in 3% (w/v) agarose before cutting. PCLS were harvested and taken into culture (37°C, 5% CO₂ atmosphere). The incubation medium was the same as the slicing medium, except for the additional supplementation of 1 mM sodium pyruvate, 2 mM glutamine, amino acids (PAA Laboratories, M11-002, 1∶50) and vitamins (PAA Laboratories, N11-002, 1∶100). For measurements, only slices with airways free of agarose, with beating cilia and an intact and relaxed ASM layer were used.

Electric field stimulation (EFS)

EFS were performed as described before for rats [21]. Briefly, a PCLS was transferred to a cavity of a standard 12-well plate, placed between two platinum electrodes, weighted down by a Teflon ring and 1 mL incubation medium was added. The electric field was delivered by a Hugo Sachs Electronics Stimulator II (March-Hugstetten, Germany). A standard electric stimulation comprised of a stimulation train lasting 3.3 min with a train rhythm of TR = 60 s, a train width of TW = 2.5 s, a frequency of F = 50 Hz, a pulse duration of B = 1 ms and a current of A = 200 mA (equal to a voltage of 40 V). Airways were monitored by videomicroscopy and used for quantification. Images were analyzed using Optimas 6.5 software (Optimas, Bothell, WA, USA). The airway area before the first stimulation was defined as 100% initial airway area (IAA).

Frequency response curve

In addition to the standard EFS protocol, EFS was conducted at increasing frequencies (0.4–100 Hz). Each frequency was applied once for 2.5 s and after a pause of one minute the next frequency was examined.

Pharmacological interventions

Neural stimulation was verified by the use of high magnesium concentrations known to block neuromuscular transmission [27], [28]. PCLS were repeatedly stimulated. The first stimulation train was carried out in the absence of magnesium sulphate. The second stimulation followed 30 min after the addition of 10 mM magnesium sulphate. Subsequent addition of 10⁻⁴ M methacholine confirmed normal ASM physiology.

Functional innervation was characterized by the use of pharmacological compounds. 10 µM neostigmine and/or 10 µM atropine, concentrations which have been shown to be effective before [21], were added before a particular EFS train was used to study parasympathetic cholinergic innervation. Exogenous addition of 10 µM capsaicin, a compromise according to two former studies [3], [29], was used to study eNANC innervation. The influence of TRP channels on eNANC responses of guinea pig PCLS was examined by the use of 10 µM ruthenium red or 30 µM SKF96365 [30].

Statistics

Non-linear regression and statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, USA) or JMP 9 (SAS Institute, Cary, NC, USA). Homoscedasticity was checked by the Bartlett test. If variances were unequal, unpaired data were either compared by the Mann-Whitney test (2 groups) or the Steel-Dwass test (>2 groups); if variances were equal by the t-test (2 groups) or analysis of variance (ANOVA, >2 groups) followed by the Tukey test. Paired data were analyzed by the paired t-test. The statistical test used is indicated in the legends of the table and figures. The median effective frequencies (EF₅₀) were calculated by four parameter logistic regression and compared by the F-test. P<0.05 was always considered significant.

General considerations on EFS of PCLS

Recently, we introduced rat EFS as a means to study neural responses in PCLS and found the following conditions to be effective [21]: F = 50 Hz, B = 1 ms, A = 200 mA and TW = 2.5 s. The same parameters were used to stimulate PCLS in the present study and evoked characteristic airway contractions in each species (figure 1), except for the mouse. The neural mediation of these responses was demonstrated by the inhibitory effect of magnesium concentrations (10 mM) high enough to prevent voltage-induced calcium entry and the release of neurotransmitters at the nerve terminals [27], [28] (table 1). We showed that the contraction to exogenous methacholine was not affected by magnesium to exclude that the high magnesium concentration alters ASM physiology itself. These findings show that under our conditions EFS acts on neurons and not directly on ASM.

Under our standard conditions (F = 50 Hz, B = 1 ms, A = 200 mA≙40 V, TW = 2.5 s) mice were completely unresponsive. However, EFS elicited contractions in tracheal preparations of mice in previous studies [32], and in line with that we observed neurally evoked bronchoconstriction in murine PCLS at higher electric fields (figure 2). In sheep PCLS, on the other hand, airways were highly excitable (figures 1, 3) and nerve activation was observed already at low frequencies or even after one single electrical stimulus (0.4 Hz at train width 2.5 s). Overall, the EF₅₀ values that we observed are in line with prior studies on bronchial or tracheal preparations [33]–[36], which demonstrates that PCLS are a suitable tool to study neural regulation of airway tone. Among all species studied here, the EF₅₀ value of guinea pig PCLS (7.0±0.5 Hz) is closest to that of humans (10.2±0.4 Hz), providing an argument for our conclusion that guinea pig airways are a reasonable proxy for human airways.

We believe that the differences between the species are not explained by differences in ASM cells, because the responses to methacholine are very similar in all species [10], [12], [13], [15], [16], [18]. Additionally, the experiments with magnesium exclude direct electric activation of ASM. Possible explanations are differences in neurotransmitter release or degrading enzymes such as the acetylcholine esterase that may vary in amount or activity [37]–[39]. However, we believe that the type of innervation, i.e., cholinergic, adrenergic, eNANC or iNANC, is the most relevant reason for the differences between species, as for instance the repetitive reversible contraction in rat is cholinergic [21], whereas the steady contraction without relaxation in guinea pigs (figure 1) suggests an additional mechanism other than a cholinergic one in which the neurotransmitter is not metabolized.

Pharmacological characterization of distal neural airway responses

The type of innervation was addressed by pharmacological agents: The muscarinic antagonist atropine was used to show the involvement of parasympathetic cholinergic nerves. The TRPV1 channel activator capsaicin as well as the unspecific TRP channel blockers SKF96365 or ruthenium red were used to address eNANC nerves.

Atropine blocked the EFS-induced bronchoconstriction in PCLS from rats, guinea pigs, sheep, marmosets and humans (figure 4), indicating that these species receive cholinergic innervation in the distal parts of their lungs. These findings extend former studies on larger airways [3], [33] and provide important new information, because innervation is unevenly distributed along the tracheobronchial tree [33]. This is also true for tachykinergic innervation in guinea pigs that before was thought to supply predominantly the large airways [33]. In this species, distal airways appear to be supplied by both cholinergic and tachykinergic nerves, as shown by the effects of atropine and the TRP channel blockers (figure 4 and 6). Moreover, in contrast to the rats, in which cholinergic responses are weaker in smaller airways [21], distal airways from sheep or human were quite susceptible to EFS-induced cholinergic bronchoconstriction (figure 4), indicating relatively strong innervation of peripheral airways in these species. The residual airway contractions in the presence of atropine may be due to eNANC activation, due to blockade of presynaptic muscarinic receptors by atropine, preventing the autoinhibitory effect of acetylcholine release [40], [41] or due to release of substance P that might potentiate the release of acetylcholine from cholinergic nerves [40].

Various types of TRP channels are expressed in the mammalian lung and are involved in the regulation of airway functions, in which particularly TRPV1 and TRPV4 play an important role. TRPV1 channels are generally expressed in non-myelinated C-fiber afferents, which innervate the airways and contain sensory neuropeptides [42]. Activation of TRPV1 channels on nerves induces calcium influx into neurons, membrane depolarization and action potentials. Such excited chemosensitive nerves correspond to eNANC nerves, which induce bronchoconstriction by the release of substance P or a related tachykinin [43]. Therefore, we studied the effects of capsaicin, a potent TRPV1 agonist [31], [42], to examine the role of eNANC innervation in the various species. Notably, only airways of guinea pigs and human responded to capsaicin providing further evidence for the presence of tachykinergic nerves in the distal lungs of these species (figure 5) and further suggesting the guinea pig as a good model for the human situation. In support of this conclusion, Undem and coworkers [29] reported contractions of 66±10% in guinea pig bronchi towards capsaicin and Spina's group [3] made similar observations in human airways (1 µM: 2±3%, 10 µM: 14±13% and with 100 µM capsaicin 44±40% of the maximal contraction by 10⁻⁴ M methacholine). Studies by Lundberg and colleagues [44] support the notion that human airways are less responsive to capsaicin than those from guinea pigs, by showing guinea pig tracheas being 1000-fold more sensitive to substance P and 100-fold more sensitive to capsaicin than human bronchi. Further, Lundberg's and colleagues' [44] study also supports our observation that not all human airways are capsaicin sensitive. From studies with transmural EFS of human segmental bronchi, they report two different scenarios: one where contractions were abolished by atropine and another one where only tetrodotoxin but not atropine was effective. These findings suggest the existence of two types of human airways: either solely innervated by cholinergic nerves or innervated by both cholinergic and eNANC nerves.

That capsaicin failed to evoke clear bronchoconstriction in PCLS from marmoset is in line with data from marmoset tracheae and bronchi [3]. These only contracted by 100 µM capsaicin up to 2.3±2.8% respectively 8.0±6.6% of the maximal contraction induced by 100 µM methacholine [3]. In rat and mouse bronchi, but probably also in marmoset and sheep airways, capsaicin may play a more prominent role in the activation of sensory nerves of the inhibitory system than in the excitatory system [45], [46]. This is especially true for the mouse, because in mouse PCLS the airway relaxed in response to capsaicin by 13±6.7% (figure 5). As contraction in sheep airways are not completely reversible under standard condition (figure 1) a capsaicin response would be expected. However, lack of full reversibility is due to incomplete recovery phase between electric stimulation. Prolonged recovery phase leads to full reversibility of sheep airways. In contrast to rat airways, the full reversibility of sheep airways within in one minute is not achieved, since first contraction of sheep airways was much stronger and second the concentration or activity of the acetylcholine esterase in the synaptic cleft may have been low. Latter argument is supported by our unpublished observation, that neostigmine fails to increase EFS-induced bronchoconstriction in sheep (mean of contractions±SEM: 50.4±12.5%-IAA w/o neostigmine vs. 71.3±5.4%-IAA plus neostigmine, n = 5, p = 0.1594 in t-test).

Neural stimulation can also cause bronchorelaxation via either sympathetic or iNANC nerves [2]. In the present study, only marmoset airways enlarged to a caliber greater than the initial caliber after multiple EFS (figure 1). Only the combination of a β-receptor antagonist (propranolol) and a nitric oxide synthase inhibitor (L-NAME) was effective to block the relaxation (data not shown) suggesting that both types of nerves are involved in regulating marmoset airway tone. Hence, the marmoset may be a useful model to study relaxant innervation of the lung, especially as no precontracting agents are needed. Whether the peripheral airways of the other species are or are not innervated by relaxant nerves has still to be elucidated, because usually relaxation becomes apparent only in precontracted airways [47].

Airway pharmacology varies strikingly between species, not only with respect to neural transmission, but also with respect to various inflammatory mediators. For instance, airways from rodents, marmosets or rhesus monkeys do respond weakly or not at all to leukotrienes [16], [48], that readily contract airways in humans [49], cynomologus, baboons [16] and guinea pigs [13]. The ready contraction of guinea pig airways by leukotrienes is not only found in PCLS, but also in tracheal or parenchymal preparations as well as in vivo [50]. In contrast, in ventilated mice leukotrienes did not induce bronchoconstriction [51]. Therefore from our observations and from the literature two conclusions can be made: first species differences observed in PCLS translate to other model systems and most likely to in vivo; second, with respect to airway pharmacology guinea pigs resemble humans more closely than do mice or rats [52], [53]. Belonging to the family of callitrichidae, the non-human primate marmoset is the closest relative to humans among the species studied [54]. Neural airway responses were similar between the marmoset and humans and we therefore consider the marmoset as a useful primate model to study airway pharmacology.

In conclusion, for the first time airway innervation was studied in various mammals including humans in the same lab and under identical conditions. PCLS represent a useful model to examine peripheral airway innervation, because nerves remain functional and can be activated specifically. Using this model we showed that the small airways of rats, guinea pigs, sheep, marmosets and human do all receive cholinergic innervations. Moreover, distal guinea pig and human airways do also receive eNANC innervations. Furthermore, it was shown that PCLS are a suitable tool to investigate physiological consequences of TRP channels in the lung. Since guinea pig and marmoset neural responses correlate with the human responses reasonably well, we consider these species suitable for studies on the role of peripheral airway innervations relevant to human lung diseases.