Department of Pediatrics and Neonatal Perinatal Research Institute, Duke University Medical Center, Durham, North Carolina
Submitted 5 January 2005 ; accepted in final form 6 June 2005
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ABSTRACT |
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ontogenesis; viscoelasticity
The internal resistance to shortening can be considered as a general concept referring to all factors internal to the contractile tissue that oppose shortening. These factors likely depend on the elastic, frictional, and viscous properties of each tissue component. They are inherent in the response of structural components of the tissue to the deformation produced by the contractile response. Stiffness, defined as change in force per unit change in length, is a measure of tissue response to geometrical deformation. We reason that the elements contributing to tissue stiffness are also likely to contribute to resistance to shortening.
In the unstimulated muscle tissue, the cytoskeleton and extracellular components are likely the major contributors to tissue stiffness. When the muscle is stimulated to contract, the formation of cross bridges determines additional and substantial structural interconnections among different cell components. The number of attached cross bridges, their relative arrangement, and the compliance of thin and thick filaments become important contributors to stiffness during a contractile response. In other words, the tissue stiffness of a contracting muscle is determined by both noncontractile and contractile factors. Moreover, shortening of smooth muscle at physiological lengths is opposed by increasing external load due to the deformation of surrounding noncontractile structures. Indeed, while isolated smooth muscle is able to shorten by extreme amounts such as 7080% of its length (25), this maximal possible capacity and velocity of shortening is usually not achieved in situ. Smooth muscle shortening is further opposed by the radial constraint exerted by the connective tissue wrapped around cells (15), which has been shown to increase muscle stiffness, especially at extreme shortening (16). Therefore, both the stiffness of noncontractile structures and the stiffness of cross bridges are relevant to the shortening velocity and capacity. We sought to evaluate the stiffness of intact tracheal strips both in the absence and at the peak response to an external stimulation to assess the contribution of the noncontractile structures in the overall muscle stiffness.
We also sought to investigate whether the stiffness of intact tracheal muscle strip changes with the stage of maturation. Intact tracheal strips used in this study contain the airway epithelium, lamina propria, basement membrane, loose connective tissue in the submucosa, smooth muscle, and interstitial connective tissue between muscle cells. Although the epithelium is unlikely to contribute to the mechanical stiffness of airway tissue (28), maturational changes in the other tissue components could form the structural basis of changes in tissue stiffness. It has been shown that extracellular matrix and lung tissue mechanics undergo maturational changes in rats (26). Mitchell et al. (17) studied pre- and postnatal development of smooth muscle cells in rat lung. They used -actin staining to identify progenitors of smooth muscle cells or differentiated smooth muscle and showed that it was more abundant in the immature lung. They also found that the intermediate filament protein, vimentin, decreases with maturation and is gradually substituted by desmin, thus suggesting substantial changes in smooth muscle cytoskeleton with maturation. These structural findings support the rationale for investigating the maturational changes in stiffness of intact tracheal strip to evaluate its potential contribution to the prominent and unique contractile features in juvenile airway smooth muscle.
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METHODS |
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Hartley guinea pigs were obtained from Charles River Laboratories (Wilmington, MA). The animals were divided into three age groups, and five from each age group were used for this study. The three age groups were (means ± SD): 1-wk-old (1-wk, age = 6.8 ± 0.8 days, wt = 130.8 ± 4.5 g), 3-wk-old (3-wk, age = 21.2 ± 0.8 days, wt = 250.4 ± 27.8 g), and 3-mo-old (adult, age = 90.0 ± 3.9 days, wt = 782.2 ± 26.8 g). All animal procedures were approved by the Duke Institutional Animal Care and Use Committee. Anesthesia was induced with pentobarbital sodium (200 mg/kg ip) and confirmed by the absence of a reflex response to toe clamping. The trachea was removed and immersed in Krebs-Henseleit (K-H) solution on ice and aerated with 95% O2-balanced CO2. The K-H solution contained (in mM) 115 NaCl, 1.38 NaH2PO4, 25 NaHCO3, 2.5 KCl, 2.46 MgSO4, 11.2 dextrose, and 1.9 CaCl2. Under a dissecting microscope (SZH10 Olympus stereomicroscope), loose connective tissue was carefully removed from outside the trachea. The length of the muscle was measured under the dissecting microscope both before (in situ length) and after (in vitro length) the cartilage rings were cut, and the trachea was opened longitudinally through the center of cartilage rings. Specifically, the in situ length was measured as the entire length of airway smooth muscle in an intact tracheal ring, i.e., from the cartilage attachment on one end to the attachment on the other end. Each tracheal strip was dissected with cartilaginous attachment on both ends. One cartilage attachment was clamped in a phosphor-bronze clip that would be later inserted to the bottom of an 80-ml water-jacketed organ bath filled with oxygenated K-H at 37°C. The other end of the cartilage attachment was tied to the tip of a transducer of the servo-controlled lever system using a 4.0 silk surgical thread. To obtain accurate stress and strain data (see Data Analysis), we took particular care in measuring the initial dimensions of the tracheal strips and used multiple measurements for validation. Micromarkers (carbon powder) were inserted on both ends of the parallel-oriented muscle fibers for monitoring the actual length of the muscle (Fig. 1) and to ensure that the length was always measured from the same segment of each tracheal strip. The initial length (L0), the initial width (wo), and the initial thickness (tho) were measured when the muscle strips were held vertically by the minimal force required to keep the strip from bending (magnitude negligible). The product of wo and tho defined the initial cross-sectional area (CSA0) of a muscle strip and was used to normalize force in each strip throughout the experiment. The length was measured using a color video camera (Hitachi VK-C370 digital signal processor with Nikon c-mount adapter) and a Sony television monitor. The dimensions projected on the television monitor were calibrated with a ruler placed adjacent to the muscle strip in the tissue bath. After the measurement of the initial length, a preload of 1 mN was applied to the transducer, and the muscle strip was allowed to equilibrate isotonically for 1 h.
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Protocols For Strip 1: Static Measurements of Stiffness at Incremental Loads and Dynamic Measurements at a Preset Load
Passive stiffness measurements. We defined passive stiffness as the change in force per unit change in length measured in unstimulated muscle strips.
Static tensile test. After equilibration, the preload was increased incrementally by 1 mN at 10-min intervals until the load reached 9 mN while the muscle length at each preload was monitored using the video camera and television monitor. The force exerted on the muscle strip was recorded and filtered through a 5-Hz digital filter to reduce noise. The load was maintained constant throughout the 10-min time interval by setting the muscle in isotonic mode. The muscle dimensions associated with each preload were measured 10 min after the load was imposed. This time duration between consecutive preloads was determined through preliminary tests that showed that this would allow sufficient time for the muscle length to reach a steady state after each stretch. Stiffness was calculated as described below in Data Analysis.
Dynamic oscillation test. The preload was reduced to 5 mN after the static tensile test. Two types of mechanical sinusoidal oscillations were applied to the muscle strip at this preset load: 1) oscillations controlling the applied load, which produced 30 and 75% change of the preset load and resulted in a length perturbation of 1 and 3% of muscle length, and 2) oscillations controlling the strip length, which produced a 4 and 8% change of muscle length. The frequency of the oscillation wave form was 0.5 Hz. The force signal and position signal of the transducer lever were recorded at a sampling frequency of 75 Hz. The resonant frequency of the force transducer was 85 Hz according to the manufacturer's specifications. The motion controller was equipped to do force feedback control. Stiffness was calculated as described below in Data Analysis.
Active stiffness measurements. Active stiffness refers to the stiffness of muscle strips while a stimulus is applied and maintained. Active stiffness was measured in the same strips as passive stiffness. After completion of the oscillation at 5-mN preset load, tension was reduced to 1 mN, and sufficient time was allowed to elapse (5060 min) for length to return to its original value. Carbachol was then added to the tissue bath and maintained at a concentration of 105 M. The muscle strip was allowed to contract isotonically against the preload of 1 mN.
Static tensile test. After the maximal shortening was achieved, the length of the muscle was measured and the preload was increased incrementally as described above for unstimulated muscle strips. Force exerted on the muscle strip and the associated length were also measured the same way as unstimulated strips.
Dynamic oscillation test. Mechanical oscillations controlling the applied load and controlling the strip length were performed as described above. The only difference in protocol from unstimulated muscle strips was that the oscillations were imposed at the highest preload achieved at the end of the static tensile test instead of 5 mN.
Protocol For Strip 2: Passive and Active Stiffness After Adaptation To a Length When Maximal Force Is Generated
After the 1-h equilibration period, the strip was set in isometric conditions, and a partial length-tension curve elicited as follows: the muscle strip was incrementally stretched and stimulated once at each intermediate length with electric field stimulation (EFS, 60 Hz, 18 V). When no increase in force due to incremental stretch was observed, the strip was not further stretched and the muscle was allowed to adapt by repeated EFS until maximal force (Fmax) was achieved (20, 27). Stiffness at this preset length was measured only by oscillations controlling the strip length. The same oscillation parameters described for strip 1 were used. Measurements were obtained at two time points (before activation and at peak activation by EFS) that reflected passive and active stiffness, respectively. Stiffness was also measured at the peak contraction to carbachol (105 M). Between sets of oscillation, the muscle was stimulated with EFS three to four times to ensure that the viability of the muscle remained unchanged. Data were not used if the force generated from EFS fell <85% of Fmax.
Data Analysis
Stress-strain data were calculated from tension (F), CSA0, the length of tissue sample (L), and the L0 following the definitions of engineering stress () and engineering strain (
) (10).
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Static tensile test.
A linear relationship ( = a + b x
; a and b are constants) was fitted to the stress-strain data. We defined stiffness as the slope (b) of the regression line. Under isotropic conditions, this slope would be considered the Young's modulus, but otherwise it is referred to as the elastic modulus or tissue stiffness.
Dynamic oscillation test.
In recent years, analyzing the oscillatory dynamics of stress and strain in the frequency domain has been considered the most accurate method to measure stiffness. In our study, 20 cycles of oscillations were analyzed in each set of experiments. The ratio of noise to signal was estimated to be 25%. For ease in evaluating the magnitude and time relationship of stress and strain obtained during oscillation, the data were transferred to the frequency domain. A computer program was written using Quick Basic language to filter stress-strain data, to convert the data into frequency domain through discrete Fourier transformation, and to obtain a transfer function, i.e., the ratio of transformed strain to transformed stress.
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Note that the transfer function in our algorithm has strain as the numerator and stress as the denominator, which is the reciprocal of the definition for elastic modulus or tissue stiffness (9). The transfer function was set up this way because by convention the output is expressed as the numerator of the transfer function (9, 24).
Mathematically, a transfer function yields, at each frequency, a complex number with a real part and an imaginary part. The amplitude ratio, or absolute gain, is the absolute value of the complex number and was calculated as
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Equipment Response
We mounted a standard spring of known spring constant in place of a tissue preparation and performed the same test protocols. The measured stiffness approximated the given spring constant. The phase angle measured was close to zero (to the 3rd decimal point). This is expected from a perfectly elastic spring, since the phase angle is zero for an ideally elastic material in which all energy is stored. This validated our approach and indicated that the phase lag within the apparatus was negligible. Therefore, our results reflected solely the tissue response.
Statistics
Stiffness measurements from different age groups and in the absence and presence of stimuli were compared using ANOVA and least significant difference (LSD) post hoc analysis. Phase-angle measurements from different age groups were compared using the Kruskal-Wallis analysis because our statistical software (Statistix 8; Analytical Software, Tallahassee, FL) revealed that the phase-angle data did not satisfy the conditions for parametric analysis. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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The entire length of the tracheal smooth muscle could be visualized under dissecting microscope while the tracheal cartilage ring was still intact. This length, i.e., the in situ length, was 1.1 ± 0.1, 1.5 ± 0.2, and 2.2 ± 0.1 mm in 1-wk, 3-wk, and adult groups, respectively. After the cartilage rings were cut, the length of the muscle from one end of the cartilage to the other was reduced by 32 ± 2% in 1-wk, 29 ± 10% in 3-wk, and 53 ± 2% in adult groups. This indicates the extent by which the muscle was in fact stretched in intact trachea and represents a length at which there was a balance between the rest tension in the muscle and the tension exerted by the cartilage ring.
Initial Dimensions of Muscle Strips
The initial dimensions (means ± SD) of the tracheal strips from 1-wk, 3-wk, and adult groups, respectively, were L0 0.8 ± 0.1, 0.9 ± 0.1, and 1.0 ± 0.2 mm, wo 1.2 ± 0.2, 1.5 ± 0.3, and 1.8 ± 0.2 mm, and tho 0.5 ± 0.1, 0.7 ± 0.1, and 0.8 ± 0.1 mm. These were the dimensions obtained after the strips were mounted in the organ bath under no load (Fig. 1) and used to convert force and length data of individual strips into stress and strain.
Passive Stiffness
Static tensile test.
The goodness of fit of the linear function ( = a + b x
) to the stress and strain data from the static tensile test of unstimulated muscle strips was expressed by R2 values (means ± SD): 0.97 ± 0.02, 0.96 ± 0.04, and 0.97 ± 0.01 in the 1-wk, 3-wk, and adult age groups, respectively. This indicates that a linear relationship was a good fit of the data within the range of strain used in this study. Representative linear regressions and the mean stiffness are shown in Fig. 2 (left). No significant age-related difference was detected.
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Static tensile test. The stress-strain relationship obtained from a static tensile test of muscle strips in 105 M carbachol was fitted to the same linear function as that used to calculate passive stiffness. As indicated by R2 values 0.97 ± 0.02 in 1-wk, 0.92 ± 0.08 in 3-wk, and 0.97 ± 0.02 in adult groups, the linear function was also a good fit for active static stiffness in all age groups. A representative linear regression and the mean stiffness are shown in Fig. 2 (right). Although the amplitude of stiffness more than tripled that without stimulation, there was no significant age-related difference detected using the static tensile test.
Dynamic oscillation test. Unlike in unstimulated strips, no statistically significant age-related difference in stiffness was found in activated muscle strips. The stiffness in carbachol-stimulated strips calculated from dynamic oscillation at preset length (strip 2) and at preset load (strip 1) is shown in Fig. 5. Similar to that found in static tensile test, the active dynamic stiffness approximately tripled that without stimulation. Stiffness at the peak response to EFS was 11.44 ± 2.65, 12.96 ± 2.67, and 18.95 ± 5.56 in 1-wk, 3wk, and adult guinea pigs, respectively.
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DISCUSSION |
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As with most soft tissues, intact tracheal strips displayed viscoelastic properties. These properties are shown by the phenomena of stress relaxation and creep, which were observed in our preliminary tracings as well as by the observation that the length of the tissue takes time to return to the same as previously when the force is removed (9). The core finding of this study is a systematic characterization of the viscoelastic property of intact tracheal strips during the course of maturation and at peak response to a contractile stimulus. This characterization is through two parameters, the stiffness and the phase angle. Stiffness was studied by analyzing the stress-strain relationship from data obtained under static tensile tests and dynamic oscillation tests. The results of this study show that the two methods, although distinctly different in experimental and analysis approaches, yielded values of tissue stiffness of the same order of magnitude. The phase relationship between stress and strain was studied through the phase angle estimated from oscillation tests. The phase angle indicates the fatness of the loop when stress data are plotted against strain data and is related to the ratio of energy dissipation to energy stored through its tangent function, i.e., hysteresivity (68, 23, 26). An angle different from zero is an indication of dissipative mechanisms, including viscous and frictional effects. Indeed, while a purely elastic material has a phase angle of zero, a purely viscous material has a phase shift of 90° (13, 24). Therefore, a viscoelastic tissue has a phase angle between 0 and 90°. Both stiffness and phase angle are dependent on the extent of the stored and dissipated energy.
No age-related difference in the viscoelastic properties was found in the presence of an external stimulus. We used two types of contractile stimuli: carbachol was chosen because it induces a stable contraction in guinea pig tracheal strips and EFS was chosen because of its physiological features. At all ages, stiffness of contracted muscle, which we defined as "active stiffness," reached a higher level than at rest. Active stiffness appeared to be related to the extent of the contraction, being greater in response to carbachol than to EFS. When muscle is stimulated to contract, cross bridges are formed. Both the cross bridges and the passive components of the tissue strip contribute to active stiffness. In fact, the net increase in stiffness due to the presence of stimulation was approximately twofold the passive stiffness, suggesting that passive stiffness constitutes a significant portion of total stiffness. Interestingly, we found a reduction of the phase angle due to stimulation in infant and adult strips but not in juvenile strips, thus abolishing the age differences observed at rest. The factors that could affect the in/out of phase response would be those affecting the viscous and frictional mechanisms. The reduced phase angle accompanying the increase in stiffness may, therefore, indicate reduced frictional and/or viscous properties due to smooth muscle contraction in infant and adult strips. This might be explained structurally by cross-bridge formation turning a more fluid-like resting tissue (viscous and frictional) into a more organized and therefore more solid-like (elastic) contracted tissue. The lack of phase-angle reduction in the juveniles could be partly due to the phase angle of juvenile strips at rest being already low. Alternatively, a structural component other than the ones responsible for the increase in stiffness, i.e., cross-bridge formation, may undergo maturational changes and be responsible for the age-dependent regulation of the viscous/frictional property in muscle tissue.
Age-related differences were mainly observed in muscle preparations that were not activated by a contractile stimulus. The oscillation test showed that the stiffness at rest is similar in infant and adult but is lower in juvenile animals. Passive stiffness reflects the distensibility of the muscle and is therefore relevant to the load that the muscle must overcome when stimulated to contract. The existence of substantial smooth muscle tone at rest in guinea pigs could contribute to passive stiffness in this species. However, this baseline activation could not account for the age difference in the viscoelastic properties studied here because the intrinsic tone in 3-wk strips is not significantly different from the other age groups (3). The different stiffness with age can therefore be ascribed only to noncontractile structures. One source of load against contracting muscle is the deformation of the airway wall. Thus whereas airway smooth muscle from trachea and main bronchi is capable of shortening in vitro by 7080% of the starting length (25), it shortens only by 30% in vivo (19). The deformation of a stiffer material requires greater force and therefore produces greater load. Cartilage provides most of the afterload impeding muscle shortening in the trachealis (21), but we designed our study to exclude the contribution of cartilage to load. Therefore, our observation that the passive stiffness of strips comprised nearly a third of the total stiffness (when maximally contracted) suggests that the soft noncontractile components of the tracheal strips are also capable of imposing a load to limit the degree of shortening in vitro. The stiffness of the airway mucosal membrane, which has been recognized to provide a mechanical load on the smooth muscle, is reported to be
324 kPa in sheep (5) and rabbit (28). The stiffness measured in guinea pig tracheal strips is of a similar magnitude. This stiffness represents the extensibility of the stress-bearing structures within the tracheal strip, which likely include the mucosal membrane, the connective tissue between smooth muscle cells, and the cytoskeleton within the cells. It is important to note that the level of stretch imposed on the strips during our experiments was chosen to be within the in situ length of the tracheal strips. The in situ stretch was greater in adult than in younger age groups. This is not surprising considering the load applied to tracheal smooth muscle originated from the deformation of the tracheal cartilage rings and that an age-related increase in cartilage stiffness has been reported in bovine articular cartilage (2). This age-related cartilage stiffening was related to the age-dependent content of enzymatic and nonenzymatic collagen cross-linking. By keeping the strips within the range of the in situ stretch, we ensured that results reflect the physiological behavior of the smooth muscle. It should be pointed out that it is within this length range that the stress-strain relationship was found to be linear. The distinct age-related difference in passive stiffness could reflect a possibility that different stress-bearing structures are at play during maturation. Both the extracellular matrix (26) and the intermediate filaments (17) have been reported to undergo maturational changes. Specifically, the intermediate filament maturation consists of changes in the relative content of constituent proteins. It has also been shown that the constituent proteins of the intermediate filaments have different viscoelastic properties (12). Moreover, stiffness is substantially reduced in lung tissue by treatment with elastase and/or collagenase (29). Although the specific ontogenetic changes in the structure of collagen or elastin and in the protein profile of intermediate filaments remain to be studied, they likely contribute to the lower stiffness of juvenile airway smooth muscle, allowing for the reduced internal resistance and faster shortening we have shown at this age.
The phase angle measured when the tracheal strips were at rest also reveals a special feature that occurred only in the juvenile group. In addition to the lower stiffness, juvenile strips showed a reduced phase angle compared with the other groups. The viscous or frictional mechanisms of the tracheal strip could be regulated by structures such as collagen and elastin fibers as well as proteoglycan and hyaluronic acid. Indeed, digestion of glycosaminoglycan increased the lung tissue hysteresivity (1). Whether changes in these structures occur during maturation and contribute to the reduced phase angle in juvenile airway smooth muscle needs to be investigated. We suggest that reduced dissipative mechanisms in the juvenile airway smooth muscle may also contribute to the increased shortening velocity and capacity at this age.
We measured the stiffness and phase angle of tracheal strips from guinea pig during the course of maturation as well as when the strips from various maturation stages were maximally activated. We found that the stiffness and phase angle were the least in juvenile animals compared with the infant and adult tracheal strips. At maximal activation, the stiffness increased in all ages and the phase angle decreased, abolishing age-related differences. This study provides evidence that maturation of passive stiffness may be responsible for the reduced resistance to shortening in juvenile airway smooth muscle strips.
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GRANTS |
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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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