Canine trachealis muscle shortening and cartilage mechanics.

Canine trachealis muscle will shorten by 70% of resting length when maximally stimulated in vitro. In contrast, trachealis muscle will shorten by only 30-40% when stimulated in vivo. To examine the possibility that an elastic load applied by the tracheal cartilage contributes to the in vivo limitation of shortening, single pairs of sonomicrometry crystals were inserted into the trachealis muscle at the level of the fifth cartilage ring in five dogs. The segment containing the crystals was then excised and mounted on a tension-testing apparatus. Points on the active length-tension curve and the passive length-tension relation of the cartilage only were determined. The preload applied to the muscle before contraction varied from 10 to 40 g (mean 21 +/- 4 g). The afterload applied by the cartilage during trachealis contraction ranged from 13 to 56 g (30 +/- 6 g). The calculated elastic afterloads were substantial and appeared to be sufficient to explain the degree of shortening observed in four of the seven rings; in the remaining three rings, the limitation of shortening was greater than would be expected from the elastic load provided by the cartilage. Additional sources of loading and/or additional mechanisms may contribute to limited in situ shortening. In summary, tracheal cartilage applies a preload and an elastic afterload to the trachealis that are substantial and contribute to the limitation of trachealis muscle shortening in vivo.     

Effect of inflation on trachealis muscle tone in canine tracheal segments in vitro

Isolated tracheal segments were studied in vitro to determine how inflation affects the length and tension of the contracted and relaxed trachealis muscle. Circumferential trachealis muscle lengths were measured from cross-sectional radiographs taken during stepwise inflation of intact 20-cm-long tracheal segments to an inflation pressure of 25 cmH2O. A tracheal length spanning two cartilage rings was then cut out and mounted in a tissue bath using clips attached at the points of muscle insertion into the cartilage. The ring was stretched open along the axis of the muscle, and the resulting forces of the relaxed and contracted muscle and the cartilage were measured. Muscle lengths and tensions during inflation of the trachea were determined by comparing pressure vs. length and force vs. length measurements. During inflation from 0 to 25 cmH2O, the circumferential length of the trachealis muscle contracted with 10(-5) M acetylcholine increased from 48 to 70% of its length of maximal active tension (Lmax), while the relaxed muscle increased from 80 to 93% Lmax. The length of the contracted muscle was maintained at a nearly constant proportion of its relaxed length at each pressure.     

Quantitative measurement of smooth muscle shortening in isolated pig trachea

Matched porcine tracheal rings were exposed to theophylline and increasing doses of carbachol in Krebs solution. Histological sections of each ring were traced and each of the following dimensions measured: the external perimeter (Pe) and external area (Ae) defined by the outer border of smooth muscle and inner surface of cartilage, and the internal perimeter (Pi) and internal area (Ai) defined by the luminal surface of the epithelium and the muscle length (L) along its outer border. Absolute wall area (WA = Ae - Ai) and relative wall area (PW = WA/Ae) were calculated. Carbachol-treated tracheal ring dimensions were compared with those of their matched theophylline-treated rings. In tracheal rings with intact cartilage, maximal smooth muscle shortening of 44% was achieved with 10(-2) M carbachol. In tracheal rings in which anterior and posterior segments of cartilage were excised, the trachealis muscle passively shortened by 20% and maximal shortening (10(-3) M carbachol) was 57%. Although Ai decreased with maximal smooth muscle shortening, there were no changes in the length of Pi or in WA. These data show that the cartilage in the porcine trachea exerts both a preload that passively stretches the trachealis muscle and an afterload that limits maximal smooth muscle shortening.     

In vivo loads on airway smooth muscle: the role of noncontractile airway structures.

The degree of airway smooth muscle contraction and shortening that occurs in vivo is modified by many factors, including those that influence the degree of muscle activation, the resting muscle length, and the loads against which the muscle contracts. Canine trachealis muscle will shorten up to 70% of starting length from optimal length in vitro but will only shorten by around 30% in vivo. This limitation of shortening may be a result of the muscle shortening against an elastic load such as could be applied by tracheal cartilage. Limitation of airway smooth muscle shortening in smaller airways may be the result of contraction against an elastic load, such as could be applied by lung parenchymal recoil. Measurement of the elastic loads applied by the tracheal cartilage to the trachealis muscle and by lung parenchymal recoil to smooth muscle of smaller airways were performed in canine preparations. In both experiments the calculated elastic loads applied by the cartilage and the parenchymal recoil explained in part the limitation of maximal active shortening and airway narrowing observed. We conclude that the elastic loads provided by surrounding structures are important in determining the degree of airway smooth muscle shortening and the resultant airway narrowing.     

In vivo and in vitro correlation of trachealis muscle contraction in dogs.

Maximal trachealis muscle shortening in vivo was compared with that in vitro in seven anesthetized dogs. In addition, the effect of graded elastic loads on the muscle was evaluated in vitro. In vivo trachealis muscle shortening, as measured using sonomicrometry, revealed maximal active shortening to be 28.8 +/- 11.7% (SD) of initial length. Trachealis muscle preparations from the same animals were studied in vitro to evaluate isometric force generation, isotonic shortening, and the effect of applying linear elastic loads to the trachealis muscle during contraction from optimal length. Maximal isotonic shortening was 66.8 +/- 8.4% of optimal length in vitro. Increasing elastic loads decreased active shortening and velocity of shortening in vitro in a hyperbolic manner. The elastic load required to decrease in vitro shortening to the extent of the shortening observed in vivo was similar to the estimated load provided by the tracheal cartilage. We conclude that decreased active shortening in vivo is primarily due to the elastic afterload provided by cartilage.     

Effect of intravenous papain on tracheal pressure-volume curves in rabbits

To investigate the effects of airway cartilage softening on tracheal mechanics, pressure-volume (PV) curves of excised tracheas were studied in 12 rabbits treated with 100 mg/kg iv papain, whereas 14 control animals received no pretreatment. The animals were killed 24 h after the injection and the excised specimens studied 24 h later. Treated tracheas exhibited decreased ability to withstand negative transmural pressures, reflected in increased collapse compliance: 6.2 +/- 2.1 vs. 2.0 +/- 0.5% peak volume (Vmax)/cmH2O means +/- SD, P less than 0.001, (Vmax = extrapolated maximal tracheal volume), increased kc (exponential constant that reflects the shape of collapse limb of the PV curve): 0.244 +/- 0.077 vs. 0.065 +/- 0.015 (P less than 0.001). The distension limb of the PV curve greater than 2.5 cmH2O transmural pressure (Ptm) was no different. Compliance between 0 and 2.5 cmH2O Ptm was increased in papain-treated rabbits: 4.97 +/- 1.73 vs. 2.30 +/- 0.31% Vmax/cmH2O (P less than 0.001). Tracheal volume, and therefore mean diameter, was decreased at 0 Ptm: 2.7 +/- 0.26 vs. 3.2 +/- 0.27 mm (P less than 0.001). We conclude that airway cartilage softening increases the compliance of the trachea at pressures less than 2.5 cmH2O Ptm.     

Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

In vitro tracheal mechanics by nuclear magnetic resonance imaging

Images of rabbit tracheal cross sections were obtained at a series of transmural pressures ranging from 22 to -95 cmH2O by use of a nuclear magnetic resonance imaging microscope. The excised, washed tracheas were immersed in a solution of phosphate-buffered saline made up in deuterium oxide (D2O, pH 7.3). The images are maps of proton density in the image slice (2.5 mm thick). All but one series of images showed a collapse process in which the trachealis muscle invaginated asymmetrically, i.e., the muscle appeared to favor one side of the cartilage ring system more than the other. The connecting tissue between the cartilage rings appeared to be more compliant than the rings themselves, thus suggesting that the tracheal lumen became corrugated at negative pressures. In the plane of a cartilage ring, the lumen appeared to remain patent at pressures as low as -95 cmH2O. However, between rings, where the tracheal wall was more compliant, the lumen appeared to be totally occluded at -53 cmH2O. Lumen areas in both the plane of the cartilage rings and in a plane between rings were measured from each series of printed images for six tracheas. These measurements, when normalized, averaged, and plotted against transmural pressure gave asymptotic logarithmic compliances (n1 in the model of Lambert et al., J. Appl. Physiol. 52: 44-56, 1982) of 1.2 +/- 0.4 and 20 +/- 7 for the interring and ring regions, respectively. These values are greater than the critical value of 0.5 (J. Appl. Physiol. 62: 2426-2435, 1987) and are thus consistent with wave speed flow limitation being possible anywhere in the trachea during forced expiration.     

Relationship of airway narrowing, compliance, and cartilage in isolated bronchial segments.

Structural components of the airway wall may act to load airway smooth muscle and restrict airway narrowing. In this study, the effect of load on airway narrowing was investigated in pig isolated bronchial segments. In some bronchi, pieces of cartilage were removed by careful dissection. Airway narrowing was produced by maximum electrical field stimulation. An endoscope was used to record lumen narrowing. The compliance of the bronchial segments was determined from the cross-sectional area of the lumen and the transmural pressure. Airway narrowing and the velocity of airway narrowing were increased in cartilage-removed airways compared with intact control bronchi. Morphometric assessment of smooth muscle length showed greater muscle shortening to acetylcholine in cartilage-removed airways than in controls. Airway narrowing was positively correlated with airway compliance. Compliance and area of cartilage were negatively correlated. These results show that airway narrowing is increased in compliant airways and that cartilage significantly loads airway smooth muscle in whole bronchi.     

Inhibitory innervation to the guinea pig trachealis muscle

The inhibitory innervation of the cervical trachea was studied in situ in anesthetized male guinea pigs. We measured effects of electrical stimulation of vagal motor and sympathetic trunk nerve fibers, during atropine, on trachealis muscle tension. Effects of direct transmural stimulation of trachealis muscle were also determined. We confirmed the dual nature of the inhibitory innervation to this muscle. Vagal motor inhibitory nerves are shown to be preganglionic. Neural transmission at the level of the ganglia is characterized by filtering of high frequency action potentials. The neurotransmitter at the myoneural junction is unidentified but is not norepinephrine. Maximal relaxation accounts for about 20-40% of maximal relaxations seen with transmural stimulation of trachealis muscle in the presence of atropine. Sympathetic trunk nerve fibers are also preganglionic. Neurotransmission at the level of the ganglia is apparently 1:1 at high-action potential frequencies. Norepinephrine released presynaptically has access to smooth muscle beta- but not alpha-receptors. Maximal adrenergic relaxations account for 60-80% of total transmural stimulation relaxations. Transmural stimulation relaxations appear to be accounted for by release of neurotransmitter from sympathetic adrenergic plus vagal nonadrenergic postganglionic nerve fibers.     

Structural deformation of the preterm trachea during acute distention and collapse.

The compliant airways of the premature neonate undergo episodic distention and collapse in response to changes in transmural pressure such as occur during spontaneous breathing, mechanical ventilation, and various therapeutic maneuvers. To identify and quantitate the effects of distending and collapsing transmural pressures on the structure of immature airways, tracheal segments from fetal rabbits, fixed at 0, +30, and -30 cm H2O transmural pressure, were examined using histologic and morphometric techniques. In comparison to control sections fixed at 0 cm H2O transmural pressure, application of distending pressures led to evagination of the posterior tracheal wall and significantly increased (P less than 0.05) cross-sectional area, antero-posterior diameter, circumference and muscle length, and decreased muscle thickness. Collapsed tracheal segments (-30 cm H2O) demonstrated invagination of the posterior wall and significantly (P less than 0.05) lower cross-sectional area, and antero-posterior diameter compared to the control segments; all the other parameters remained relatively unchanged. These data demonstrate extreme changes in tracheal geometry in response to the acute application of transmural pressure. From a methodological perspective, these observations suggest that fixation pressures may present significant artifact in histological analyses. Functionally, the noted deformation may lead to alterations in anatomic dead space and airway resistance, and mechanical function of the airways; all of which may compromise respiratory status in ventilated premature infant.     

Effect of tracheal smooth muscle tone on collapsibility of immature airways

To test the influence of smooth muscle tone on extremely immature airways, tracheal segments (n = 19) were excised from premature lambs at 114-121 days gestation and mounted in a chamber filled with Krebs solution. Inflation (Si) and collapsing (Sc) compliance were determined by altering transmural pressure from 30 to 0 Torr and -30 to 0 Torr, respectively, both during control (C) and after acetylcholine (ACh) administration (experimental, E). Flow (V = 2-15 l/min) was then introduced through the tracheal lumen while chamber pressure (Pc) was increased from 0 to 30 Torr and driving pressure (Pd) was recorded for both C and E conditions. Tracheae were found to be extremely compliant; both Si and Sc were significantly (P less than 0.005) lower after ACh administration. Resistance to airflow (R = Pd/V) was also significantly (P less than 0.05) lower after ACh administration at each compressive pressure and each flow value. These results suggest that the highly compliant preterm trachea exhibits pressure-flow characteristics similar to a Starling resistor, and the effects of compressive pressures may be attenuated by ACh-induced smooth muscle contraction. Comparison of these results with data from adult and newborn animals suggests a developmental difference in tracheal mechanics and pressure-flow relationships, as well as in the way airway function is altered by smooth muscle stimulation.     

Methacholine causes reflex bronchoconstriction

To determine whether methacholine causes vagally mediated reflexconstriction of airway smooth muscle, we administered methacholine tosheep either via the bronchial artery or as an aerosol via tracheostomyinto the lower airways. We then measured the contraction of anisolated, in situ segment of trachealis smooth muscle and determinedthe effect of vagotomy on the trachealis response. Administeringmethacholine to the subcarinal airways via the bronchial artery(0.5-10.0 µg/ml) caused dose-dependent bronchoconstriction andcontraction of the tracheal segment. At the highest methacholine concentration delivered, trachealis smooth muscle tension increased anaverage of 186% over baseline. Aerosolized methacholine (5-7 breaths of 100 mg/ml) increased trachealis tension by 58% and airwaysresistance by 183%. As the bronchial circulation in the sheep does notsupply the trachea, we postulated that the trachealis contraction wascaused by a reflex response to methacholine in the lower airways.Bilateral vagotomy essentially eliminated the trachealis response andthe airways resistance change after lower airways challenge (either viathe bronchial artery or via aerosol) with methacholine. We concludethat 1) methacholine causes asubstantial reflex contraction of airway smooth muscle and2) the assumption may not be validthat a response to methacholine in humans or experimental animalsrepresents solely the direct effect on smooth muscle.

     

Anisotropic material behaviours of soft tissues in human trachea: an experimental study

BackgroundHuman trachea is a multi-component structure composed of cartilage, trachealis muscle, mucosa and submucosa membrane and adventitial membrane. Its mechanical properties are essential for an accurate prediction of tracheal deformation, which has a significant clinic relevance. Efforts have been made in quantifying the material behaviour of tracheal cartilage and trachealis muscle. However, the material behaviours of other components have been least investigated.MethodsThree human cadaveric trachea specimens were used in this study. Trachealis muscle, mucosa and submucosa membrane and adventitia membrane were excised to perform the uniaxial test in axial and circumferential directions. In total, 72 tissue strips were prepared and tested. Tangent modulus was used to quantified the stiffness of each tissue strip at various stretch levels.ResultsThe obtained results indicated that all types of tracheal soft tissues were highly non-linear and anisotropic. Trachealis muscle in the circumferential direction had the most excellent extensibility; and the adventitial collagen membrane in the circumferential direction was the stiffest.ConclusionThis study is helpful in understanding the material behaviour of trachea. Obtained results can be used for computational and analytic modelling to quantify the tracheal deformation.     

Release of prostaglandin-like material from isolated cat tracheal muscle by electrical and mechanical stimulation.

Release of PGE-like material has been studied on the isolated continuously-superfused cat tracheal muscle using dynamic bioassay methods. The effluent of transmural electrically-stimulated cat tracheal muscle induced a contraction when superfused over the rat stomach fundus strip. This response did not alter with atropine, methysergide, phentolamine and propranolol but was inhibited by aspirin and Sc 19220. The same myotropic activity in the effluent was found when trachea was mechanically stimulated by an additional increase in tension. The effluent from mechanically- and electrically-stimulated tracheal muscle caused a definite relaxation when superfused over a second cat tracheal muscle contracted by serotonin and pretreated with propranolol. Electrically-stimulated cat trachea itself gave a relaxant response which was blocked by propranolol but potentiated by aspirin. From these results it was concluded that both electrical and mechanical stimulation can elicit a release of PGE-like material from isolated cat tracheal muscle.     

In vivo estimation of tracheal distensibility and hysteresis in normal adults

We used the acoustic reflection technique to measure the cross-sectional area of tracheal and bronchial airway segments of eight healthy adults. We measured airway area during a slow continuous expiration from total lung capacity (TLC) to residual volume (RV) and during inspiration back to TLC. Lung volume and esophageal pressure were monitored continuously during this quasi-static, double vital capacity maneuver. We found that 1) the area of tracheal and bronchial segments increases with increasing lung volume and transpulmonary pressure, 2) the trachea and bronchi exhibit a variable degree of hysteresis, which may be greater or less than that of the lung parenchyma, 3) extrathoracic and intrathoracic tracheal segments behaved as if they were subjected to similar transmural pressure and had similar elastic properties, and 4) specific compliance (means +/- SE) for the intrathoracic and bronchial segments, calculated with the assumption that transmural pressure is equal to the transpulmonary pressure, was significantly (P less than 0.05) smaller for the intrathoracic segment than for the bronchial segment: (2.1 +/- 2.0) X 10(-3) cmH2O-1 vs. (9.1 +/- 2.1) X 10(-3) cmH2O-1. Direct measurements of airway area using acoustic reflections are in good agreement with previous estimates of airway distensibility in vivo, obtained by radiography or endoscopy.     

Isovolumetric and isobaric rabbit tracheal contraction in vitro

Isovolumetric and isobaric tracheal smooth muscle (TSM) contraction were studied in vitro in a preparation of the whole rabbit trachea. Eight tracheae from New Zealand White rabbits were excised and mounted at a fixed length in an organ bath. Electrical field stimulation (EFS) was performed in isovolumetric and isobaric conditions at varying transmural pressures (TMP). Supramaximal stimulation with methacholine was done at 0 TMP. Active change in pressure (delta P) with EFS showed a peak at 3.1 +/- 1.06 cmH2O TMP during inflation and at 4.1 +/- 1.18 cmH2O TMP during deflation (mean +/- SE). Active delta P decreased at higher or lower TMP. Active change in volume with EFS showed a peak at 3.2 +/- 1.26 cmH2O TMP during inflation and at 1.8 +/- 0.98 cmH2O TMP during deflation. A decrease in response was also observed at higher and lower TMP. From these data, we concluded that TSM is at optimal length (Lmax) at TMP of 2-3 cmH2O. Maximal TSM shortening with supramaximal stimulation with methacholine was 32% Lmax. This figure is considerably smaller than the 80% shortening found in unloaded strips of TSM. We conclude that rabbit TSM length is close to Lmax at TMP similar to those found at functional residual capacity and that the loads that the muscle has to overcome probably contribute to the limited shortening observed in situ.     

Changes in tracheal cross-sectional area during Mueller and Valsalva maneuvers in humans

Pressure-area behavior of the excised trachea is well documented, but little is known of tracheal compliance in vivo. Extratracheal tissue pressures are not directly measurable, but transmural pressure for the intrathoracic trachea is inferred from intra-airway and pleural pressure differences. Extramural pressure of the cervical trachea is assumed to be atmospheric. The difference in transmural pressure between the intra- and extrathoracic tracheal segments should be exaggerated during Mueller and Valsalva maneuvers. We used the acoustic reflection technique to measure tracheal areas above and below the thoracic inlet during these isovolume-pressure maneuvers. We found that 10 cmH2O positive pressure increased tracheal area in the extrathoracic segment by 34 +/- 16% (mean +/- SD) and in the intrathoracic segment by 35 +/- 15%. There was a reduction in area of 27 +/- 16 and 24 +/- 14%, respectively, for the extra- and intrathoracic segments with 10 cmH2O negative pressure. We conclude that the effective transmural pressure gradients do not vary significantly between intra- and extrathoracic tracheal segments.     

Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs.

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0-20 cmH(2)O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls (P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi (P < 0.01) and smooth muscle strips (P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.     

The transmembrane protein TMEM16A is required for normal development of the murine trachea

Pathological collapsibility of the upper airways, caused by many different genetic and environmental insults, is known as tracheomalacia in humans. We determined that Tmem16a, a member of an evolutionarily conserved family of predicted transmembrane proteins, is expressed in the developing trachea. We report that all mice homozygous for a null allele of Tmem16a died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea. In addition, the development of the trachealis muscle that spans the dorsal aspect of the trachea was abnormal in Tmem16a mutants. Since the chondrogenic mesenchyme does not express Tmem16a at any time, we propose that the cartilage ring defect observed in Tmem16a mutants is secondary to an expansion of the embryonic trachea that might result from improper stratification of the embryonic tracheal epithelium or the abnormal trachealis muscle. Our data identify Tmem16a as a novel regulator of epithelial and smooth muscle cell organization in murine development. This mutant, the first knockout of a vertebrate TMEM16 family member, provides a mouse model of tracheomalacia.