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University of British Columbia McDonald Research Laboratories and iCAPTURE Center, St. Paul's Hospital, Vancouver, British Columbia, Canada V6T 1Z3
Submitted 9 December 2002 ; accepted in final form 25 October 2003
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ABSTRACT |
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elastic loads; sonomicrometry
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METHODS |
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All in vitro studies were performed with the preparation immersed in oxygenated Krebs-Henseleit solution consisting of (in g/l) 6.9 NaCl, 0.35 KCl, 0.28 CaCl2·H2O, 0.29 MgSO4·7 H2O, 0.16 KH2PO4, 2.1 NaHCO3, and 2.0 glucose, pH 7.4. Krebs solution was freshly prepared on the morning of each experiment and heated to 36°C in an organ bath.
In Vivo Measurement
The study was improved by the University of British Columbia's animal experimentation ethics committee. Five mongrel dogs were studied. Animals were anesthetized with thiopental sodium (10 mg/kg iv) and maintained with 
-chloralose (80–130 mg/kg). Animals were then intubated with a cuffed no. 8 endotracheal tube, placed in the supine position, and mechanically ventilated (15 breaths/min, tidal volume 15 ml/kg) with pure O2. Supplemental -chloralose was administered during the experiment as required.
A midline vertical neck incision was made, and the trachea was exposed from the 5th to the 15th ring. The cervical vagus nerves were identified by dissection and cut bilaterally. Connective tissue around the trachea was gently dissected away, and the posterior wall, now separated from the esophagus, was exposed by partial eversion of the trachea. The trachealis muscle was exposed by blunt dissection through the covering connective tissue. A small slit was made in the muscle, parallel to the direction of the muscle fibers, close to the insertion of the muscle into the fifth tracheal ring. A piezoelectric transducer (1 mm diameter) was inserted into the slit and secured by a purse-string suture. A second crystal was inserted 4–6 mm from the first, in approximately the same plane, on the contralateral side of the same tracheal cartilage ring. In one animal, further pairs of crystals were inserted at the 10th and 15th tracheal rings. The crystals were connected by fine wires to a four-channel sonomicrometer (model 120, Triton Technology, San Diego, CA). The sonomicrometer provided a direct-current output proportional to the length between the crystals by measuring the transit time of the ultrasonic waves from one crystal to another, as previously described (3). The sampling rate of this technique is 1,537 Hz and, therefore, easily capable of reliable measurement of changes in trachealis muscle length. After insertion of the crystals, the trachea was returned to its original orientation in the neck. Throughout the subsequent experiments, the position (wave-form) of the crystals was monitored using an oscilloscope (model 1421, Gould) to detect any changes in the orientation of the crystals that could lead to errors in measurement of length changes by variation in the leading wave.
At 30 min after crystal insertion, the distance between the two crystals at each site was recorded at functional residual capacity (FRC). FRC was defined as the lung volume obtained when the ventilator was disconnected at end expiration, and this corresponded to zero airway pressure. The tracheal segments containing the crystals were then removed en bloc, and the dog was killed by barbiturate overdose (100 mg/kg iv), confirmed by cessation of the ECG signal. The tracheal segment was then placed in an organ bath containing 36°C oxygenated Krebs solution.
In Vitro Measurement
The rings containing the crystals were dissected from the surrounding rings and mounted on a tension-testing apparatus designed for this study (Fig. 1). The ring adjacent and inferior to the rings containing the crystals was also dissected free and placed in 10% formalin for later morphometric analysis of muscle cross-sectional area.
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Tension Testing
The tension-testing apparatus consisted of a micrometer screw (range 25 mm) mounted horizontally through two blocks of high-density polyethylene (3 x 12 x 6.5 cm; Fig. 1). A vertically oriented 22-cm-long stainless steel bar was attached to the inner side of each block. A pin, which rested at its other end on the diaphragm of a pressure-sensitive transducer (±400 mmHg; model 800-100, Bently Trantect), was attached at the midpoint of one of these bars. Metal plates, which served as the attachment devices for the tracheal ring to the apparatus, were attached at the inferior end of each bar. These attachment plates incorporated a horizontally orientated roller bar onto which the cartilage ring was attached. These bars were free to rotate within their sockets in the attachment plate. Movement of the micrometer thus resulted in changes in bar and cartilage tip separation. Tension generated by this movement or by bar movement secondary to muscle contraction after stimulation by agonists was detected as alterations in pressure on the diaphragm of the pressure transducer and recorded as changes in tension. Tension changes were recorded by connection of the transducer to the same eight-channel recording device used for recording crystal movement.
Calibration
When a cartilage-muscle ring was attached to the roller bars, any tension generated by separation of the bars was, in part, the result of true alteration in cartilage tip separation. Because the bars were not perfectly rigid, there was some degree of bar bending for any tension generated. To calculate the degree of bar bending that would occur with known tensions, a rigid plastic calibration bar was placed between the two roller bars. The micrometer was moved 0.5 mm, and the resultant tension was recorded. This allowed calculation of the degree of bar movement that would occur with any generated tension. The length between the two roller bars was calibrated before each experiment by attachment of a calibration bar of known length between the roller bars and relation of this length to the reading from the micrometer dial.
Force was calibrated by attachment of a Y-shaped piece of silk thread from the roller bars and adjustment of the length between the bars until the angle between the arms of the Y at their connection with the vertical thread was 90°. Weights (10–50 g) were hung from the free end of the vertical limb of the thread, and the recording trace was adjusted until each 1-mm deflection on the paper trace represented 2 g of weight. Addition and removal of weights indicated that there was no hysteresis of the force measurements.
Mounting of Tracheal Rings
Tracheal rings were attached to the roller bars by means of 2-0 Vicryl suture material. The suture needle was first placed through a cartilage tip near the muscle insertion and then around a roller bar before it was passed again through the cartilage tip immediately adjacent to the first needle pass. The contralateral cartilage tip was attached to the second attachment plate in a similar manner. The attachment plates were then mounted onto their sites at the inferior end of the steel bars so that the trachealis muscle was positioned superiorly (Fig. 1). In this manner, stepwise changes in the separation between the two bars and, thus, the two cartilage tips could be achieved by movement of the micrometer screw.
Protocol
Passive length-tension relations of the muscle-cartilage preparation. After the cartilage-muscle ring was attached to the testing apparatus, the ring was submerged in 36°C oxygenated Krebs solution. The cartilage tips were separated until the separation of the crystals was the same as that obtained at FRC in vivo. From this point, designated zero tension and resting length, 0.5-mm incremental changes in micrometer readings were made, with approximation of the tips designated compression and separation of the cartilage tips designated extension. Cartilage tips were first compressed in a stepwise fashion using 12–16 steps (Fig. 2A). The total time for each compression was 
20 s. The preparation was then rapidly returned to resting length by continuous winding of the micrometer. The compressive steps were then repeated, and two extension experiments were performed in the same manner (Fig. 2B). During the extension experiments, only four 0.5-mm steps were performed; as in pilot experiments, we found that the preparation would not return to zero tension if greater extension was used.
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Active shortening of the cartilage-muscle preparation. When the preparation was at resting length and zero tension, carbachol (10-3 M final bath concentration) was added to the organ bath (Fig. 2C). This resulted in an increase (T in Fig. 2) in tension and a degree of muscle shortening secondary to bending of the bars.
Once the tension had reached a plateau and no further change in crystal separation was evident, the unit was returned to zero tension (Fig. 2D) by approximation of the cartilage tips using the micrometer screw, and the new length was noted. The muscle was then cut between the crystals, producing an outward recoil of the cartilage tips (Fig. 2E). Although no crystal separation record was obtainable, the resultant change in tension was recorded on the trace from the pressure transducer included in the tension-testing apparatus.
Passive length-tension relation of cartilage only. The preparation was returned to the precarbachol starting length (Fig. 2F), and the residual tension was noted (Fig. 2G). Compression and extension steps described in Passive length-tension relations of the muscle-cartilage preparation were repeated (Fig. 2H).
Morphometry. The ring immediately inferior to the study ring was placed in 10% formalin for calculation of muscle cross-sectional area. The unit was dehydrated with ethyl alcohol and embedded using glycol methacrylate (JB-4, Polysciences, Warrington, PA). Sections perpendicular to the long axis of the trachealis muscle were measured using a digital trace from microscopic enlargements, and a mean value for cross-sectional area of the trachealis muscle was calculated.
Analysis
Passive length-tension relations of the muscle-cartilage preparation. The tension generated with each stepwise decrease and increase in cartilage tip separation was calculated. The actual length change at the cartilage tips could be calculated by subtracting from 0.5 the product of the tension generated with that single step and the calibration factor for the movement of the vertical bars. Plots of changes in length against changes in tension were constructed (Fig. 3, curve a). In addition, the changes in muscle length measured with the crystals were recorded.
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Active shortening and tension. The addition of carbachol caused tension development and shortening (Fig. 3, b). The maximal tension produced by this quasi-isometric contraction was noted, and the length change between the rings and between the crystals required to return the preparation to zero tension was recorded (Fig. 3, c). The muscle was then cut, and the outward recoil of the cartilage was recorded (Fig. 3, d).
Passive length-tension relation of cartilage only. The tension generated by the cartilage at resting length was obtained as the tension recorded when the cartilage only was returned to the precarbachol starting length (Fig. 3, e). The changes in tension as a result of changes in length of the cartilage only were also plotted (Fig. 3, f). The distance shown as g in Fig. 3 equals the distance e and represents the preload that is applied to the posterior membrane before smooth muscle contraction at resting length. The distance shown as h in Fig. 3 represents the afterload provided by the cartilage after muscle contraction.
All tensions were converted to stress by dividing by the cross-sectional area of the tissue. The average cross-sectional area of the posterior membranous tissue and the portion occupied by tracheal muscle was calculated from the adjacent tracheal rings. For calculation of passive stress, we assumed that the stress was evenly distributed over the entire cross-sectional area of the posterior membrane. For calculation of active stress, we used the cross-sectional area of the trachealis muscle in the posterior membrane on the assumption that the entire active stress was borne solely by the muscle.
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RESULTS |
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DISCUSSION |
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These results, which focus on the trachealis muscle, support the studies of Gunst and Lai-Fook (4) in which measurements of airway narrowing in whole dog lungs were submaximal in vivo. Gunst and Lai-Fook postulated a number of mechanisms that could lead to incomplete smooth muscle shortening in situ: 1) trachealis muscle may be operating at lengths other than optimal length, 2) there may be incomplete activation of the smooth muscle in vivo, 3) the repeated stretching provided by ventilation may impede smooth muscle shortening, 4) neural or humeral inhibitory mechanisms may be invoked that counteract the smooth muscle contraction, or 5) loads provided by the interdependence of lung parenchyma and airway may impede the degree to which smooth muscle can shorten. An additional mechanism that could contribute to the discrepancy between in vivo and in vitro airway smooth muscle shortening is the phenomenon of airway smooth muscle plasticity. Gunst et al. (5), as well as Pratusevich et al. (11), demonstrated that airway smooth muscle shows plasticity of its length-tension relation. These investigators showed that the airway smooth muscle length-tension relation is adaptable; smooth muscle held at longer length by cartilage recoil in vivo may not be able to shorten as much as it can in vitro, because the contractile apparatus plastically adapts to the longer length. The most important mechanism that operates to attenuate the shortening of airway smooth muscle in vivo is undoubtedly related to cross-bridge dynamics and the perturbation of cross bridges that occurs because of the cyclic smooth muscle stretch that accompanies breathing (1, 11).
Although clearly important, this mechanism cannot account for the discrepancy that we have observed between trachealis shortening in vivo and in vitro, because in our in vivo experiments we suspend respiration during the isotonic contraction.
In the present study, we tried to quantitate the preload and the afterload provided by tracheal cartilage and scale it to the maximal forces that trachealis muscle was able to generate so that we could provide a reasonable estimate of the possible importance of these loads. To do this, we had to ensure that the starting length of the muscle in our in situ preparation was similar to the in vivo resting length of the trachealis muscle. To ensure this, we used the method of sonomicrometry. The sonomicrometer crystals were inserted into the trachealis muscle after sectioning of the vagal nerves bilaterally so that the measured in vivo and in situ length represented trachealis muscle length in the absence of cholinergic tone. After the cartilage ring containing the crystals was dissected out and mounted in an organ bath, there was usually some degree of muscle shortening, as reflected in the shorter distance between the crystals. With time in the organ bath, the muscle relaxed so that the in situ length could be reproduced, and it was only then that we began the measurements of passive and active length and tension. We are therefore confident that the resting length in the in situ preparation was similar, if not identical, to in vivo resting, postvagotomy muscle length. Ensuring that in situ length was similar to in vivo length does not ensure that in situ length is at the optimal length for muscle tension generation. However, in our previous studies, we showed that maximal trachealis muscle shortening in vivo occurred at FRC, and we argued that this length should represent optimal length or length very close to optimal length (9). Thus, although in our present preparation we cannot be sure that the muscle's starting length represents optimal length, we believe that it must be close to optimal length. Our previous findings using maximal concentrations of acetylcholine would suggest that incomplete activation of the trachealis muscle is unlikely to be the explanation for the limitation of shortening in vivo. Although neural inhibitory mechanisms such as the nonadrenergic noncholinergic system could potentially play a role in limiting muscle shortening in vivo, such a system has not been described in the dog and, thus, is unlikely to account for our findings. Additional inhibitory mechanisms could be release of a bronchodilating substance (e.g., nitric oxide) from airway epithelium or vascular endothelium. In the trachealis muscle, there is no interdependence between parenchyma and the airway, because the muscle is largely extrathoracic. Because we performed this study in vitro, the effect of the periodic strain related to ventilation was absent. In extrathoracic trachealis muscle, there is likely to be little or no ventilatory lengthening of the smooth muscle.
In our previous studies in which we demonstrated incomplete shortening of canine trachealis in vivo, we postulated that the tracheal cartilage rings could provide a preload, stretching airway smooth muscle to near optimal length, and an afterload, inhibiting maximal shortening (9). The mechanical properties of the tracheal cartilage have not been extensively studied, although Gunst and Lai-Fook (4) showed that the cartilage rings in dog trachea provide a stretch on airway smooth muscle in situ and that a force was required to deform the cartilage rings.
We measured different values for fractional shortening with the sonomicrometry crystals situated in the body of the trachealis muscle and by approximating the two cartilage tips using our apparatus. How could there be different fractional shortening, because the whole trachealis muscle is in series? The likely explanation is that the insertion of our roller bars onto the cartilage tips was distal to the actual insertion of the muscle into the cartilage. We sewed the roller bars onto the cartilage just external to the muscle, and we believe that the portion of the trachealis muscle that overlies the cartilage does not shorten to the same extent during contraction as does the free portion of the muscle between the cartilage tips. Different degrees of fractional muscle shortening in series have been shown in the skeletal muscle, with, in general, the greatest fractional shortening in the body of the muscle and less shortening near the tendons and insertions (4).
In an attempt to estimate the importance of the loads provided by tracheal cartilage in potentially attenuating airway smooth muscle shortening, we scaled them to the theoretical maximal capacity of the muscle (Fig. 5). The schematic length-tension diagram is based on the relations of canine trachealis isometric tension and length and isotonic shortening and length described by Stephens and van Niekerk (12). They found that a maximal isometric stress of 1.5 kg/cm2 was achieved by canine trachealis in response to cholinergic stimulation. The tension generated by each preparation was divided by the muscle cross-sectional area in that preparation and expressed as a percentage of this value.
To convert tension in our in situ preparation to stress, we divided passive and active forces by the cross-sectional area over which they applied. For calculation of passive stress, we made the assumption that the stress would be uniformly distributed over the posterior membranous portion of the trachea, which consists largely of noncontractile tissue elements. Only 10–20% of the cross-sectional area is smooth muscle (14). This is probably simplistic, and it is possible that, even under resting conditions, a greater percentage of the tension is borne by the muscle or, alternatively, by parallel elastic components. After activation of the muscle, we made the assumption that the entire increase in stress would be borne by the contractile elements. The parallel elastic elements would shorten along their passive length-tension curve, supporting less stress as shortening developed, and so we calculated active stress as the difference between the developed tension and the passive tension of the posterior membrane at the postcontracted length, which was determined by subtraction of the cartilage length-tension relation from the total tissue length-tension relation. It is likely that the parallel elastic elements that bear stress at long lengths undergo compression at short lengths, providing an additional elastic afterload to the muscle.
It is apparent from Fig. 5 that for preparations 1, 2, 3, and 6 the degree of shortening that we observed could be entirely explained by the elastic load provided by the cartilage; that is, when plotted on a length-stress curve, the end points of the curves fell close to the line that would be predicted from the preloaded isotonic curve. With preparations 4, 5, and 7, however, the elastic load provided by cartilage does not appear sufficient to explain the limited shortening we observed.
Although some of this discrepancy could be due to decreased contractility of the in vitro preparations, an additional factor may be that other elastic loads, or additional neural-humeral mechanisms, contribute to the limited smooth muscle shortening. Lambert (7) proposed that the airway basement membrane and lamina reticulars, which are elastic structures, have the potential to be load bearing, with the load increasing as the number and depth of mucosal folds increase. This load, which was not quantified in this study, could contribute to the discrepancy in some rings between measured muscle shortening and the predicted smooth muscle load.
In summary, the present study has shown that the tracheal cartilage applies a preload and an elastic afterload to the trachealis muscle that are substantial and contribute to the limitation of trachealis muscle shortening in vivo. These results suggest that changes in airway cartilage mechanics that could occur with aging or as a result of disease states could alter the ability of airway smooth muscle to narrow the airway.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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