Mechanics, Nonlinearity, and Failure Strength of Lung Tissue Ch 1

Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling ABSTRACT Enlargement of the respiratory airspaces is associated with the breakdown and re-organization of the connective tissue fiber network during the development of pulmonary emphysema. In this study, a mouse (C57BL/6) model of emphysema was developed by direct instillation of 1.2 IU of porcine pancreatic elastase (PPE) and compared with control mice treated with saline. The PPE-treatment caused 95% alveolar enlargement (P = 0.001) associated with a 29% lower elastance along the quasi-static pressure-volume curves (P < 0.001). Respiratory mechanics were measured at several positive end-expiratory pressures in the closed chest condition. The dynamic tissue elastance was 19% lower (P < 0.001), hysteresivity was 9% higher (P < 0.05), and harmonic distortion, a measure of collagen-related dynamic nonlinearity, was 33% higher in the PPE-treated group (P < 0.001). Whole lung hydroxyproline content, which represents the total collagen content, was 48% higher (P < 0.01), and α-elastin content was 13% lower (P = 0.16) in the PPE-treated group. There was no significant difference in airway resistance (P = 0.7). The failure stress at which isolated parenchymal tissues break during stretching was 40% lower in the PPE-treated mice (P = 0.002). These findings suggest that following elastolytic injury, abnormal collagen remodeling may play a significant role in all aspects of lung functional changes leading to progressive emphysema. INTRODUCTION Pulmonary emphysema is characterized by permanent destruction of the respiratory bronchioles, alveolar ducts, and alveolar walls, which leads to hyperexpansion and loss of elastic recoil (1). The most widely accepted hypothesis of how tissue destruction occurs in emphysema is that an imbalance of protease and anti-protease activity exists within the lung that ultimately leads to enzymatic degradation of elastin (38, 45). However, changes consistent with emphysema can also result from abnormality of the collagen matrix (4, 8, 12, 21, 23, 25, 35, 49). Recent studies have provided evidence that significant remodeling of the extracellular matrix (ECM) including collagen, elastin, and proteoglycans occurs during the development of emphysema both in humans (4, 12, 48, 49) and in experimental rodent models (12, 21, 25, 52). Therefore, besides elastin degradation, the biological breakdown and subsequent remodeling of collagen during the abnormal repair of the lung tissue would play a role both in the progress of this disease (24, 52) and the physiological functioning of the lung (21, 44). Among the constituents of the ECM components, elastin and collagen appear to account for most of the viscoelastic mechanical properties of the lung tissue strips (33, 46, 53). Elastic fibers behave more linearly than collagen fibers (28, 36). Therefore, one would predict that the nonlinear behavior of the lung could change when alterations occur in the relative amounts of elastin and collagen in the connective tissue. We hypothesized that if alterations occur in the collagen fiber network during the development and progress of emphysema, then both the nonlinear viscoelastic properties of the lung tissue and the strength of alveolar walls should change. To test this hypothesis, we measured the nonlinear and viscoelastic properties of the whole lung in a mouse model of emphysema induced by direct instillation of porcine pancreatic elastase (PPE) (26). To characterize lung elasticity and airway function, we measured the quasi-static pressure-volume (P-V) curves and the forced oscillatory impedance of the respiratory system. We calculated a dynamic nonlinearity index, called harmonic distortion (42), as well as measured the failure stress of parenchymal tissue strips. Finally, to assess changes in composition, we also determined the total amounts of elastin and collagen in the lung. METHODS Animal Preparation Two groups of male C57BL/6 mice (Charles River, Boston, MA), weighing 23 - 25 g were studied. Animals were treated with direct instillation of either 1.2 IU porcine pancreatic elastase (PPE) (Sigma, St. Louis, MO) dissolved in 90 µl of sterile saline (n = 18) or the same amount of saline (control) (n = 18) (26). Three weeks after the treatment, the following experiments were performed. All animal procedures were approved by the Animal Care and Use Committees of Boston University and Harvard Medical School. Measurement of Respiratory Mechanics The detailed methods are described elsewhere (20). The control (n = 8) and PPE-treated (n = 9) mice were deeply anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg), tracheostomized, and cannulated in the supine position. The cannula was connected to a computer-controlled ventilator (Flexivent, SCIREQ, Montreal, Canada). Mice were mechanically ventilated with room air using a tidal volume of 8 ml/kg at a frequency of 240 breaths/min. After stabilization, the quasi-static pressure-volume (P-V) curve of the respiratory system was measured as follows. After inflation of the lungs to total lung capacity, defined as a tracheal pressure of 25 cmH2O, mice were mechanically ventilated under the same condition for 5 sec. Following 3 sec of delay, slow volume inflation (0.1 ml/sec, total 1.2 ml) starting from end-expiratory lung volume was performed using the computer-controlled ventilator without applying positive end-expiratory pressure (PEEP) (21). Dynamic respiratory mechanics were measured at four different PEEP levels (0, 3, 6, and 9 cmH2O) in the closed chest condition and assessed by measuring impedance data during forced oscillations. To standardize volume history, each measurement was preceded by two consecutive inflations of the lungs to total lung capacity. Impedance Measurements Impedance data collection was made by interrupting mechanical ventilation for 6 s using the optimal ventilation waveform (OVW), which is a broad-band waveform containing energy from 0.5 to 15 Hz as described previously (27). The frequencies in the OVW are selected according to a non-sum non-difference criterion, which eliminates harmonic distortion and minimizes cross talk among the frequencies that are present in the input flow waveform and hence provides smooth estimates of the input impedance (43). The volumes delivered are similar to normal spontaneous tidal volume values and hence the method provides information on the mechanical properties during conditions mimicking breathing. In our experiment, we matched the peak-to-peak OVW amplitude to the tidal volume delivered by the mechanical ventilator. The ventilator displacement and cylinder pressure signals were low-pass filtered at 30 Hz and sampled at 256 Hz. With the use of Fourier analysis, impedance spectra were calculated on overlapping blocks of pressure and flow data as the ratio of the cross-power spectrum of pressure and flow and the autopower spectrum of flow. The forced-oscillatory system was calibrated by measuring the input impedance of known analogs including tubes and bottles with known impedances. The frequency response of the system was obtained, and the measured impedance spectra were off-line corrected for any phase difference between pressure and flow. Additionally, the flow-dependent impedance of the tracheal cannula was characterized separately and removed from the respiratory impedance of the mice (20). Mathematical Modeling Data were analyzed with a model that allows for specific alterations in the lung tissue similar to what might be expected in emphysema. Specifically, we applied a heterogeneous tissue elastance model of the lung (20). Briefly, in this model, we represented the airway tree by a set of airway pathways arranged in parallel, where each compartment is composed of an airway resistance (Raw), an inertance (Iaw), and a linear tissue impedance (ZLti) connected in series. Hantos et al. (16) introduced the constant-phase model and described the ZLti as: ZLti (ωn) = (G – jH)/ωnα, with α = 2/π arctan (H/G) and ωn = ω/ω0 (Eq. 1) where ωn and ω are the normalized and absolute circular frequency, G and H are the coefficients of tissue damping and elastance, respectively, j is the imaginary unit, and the exponent α describes the frequency dependence of tissue resistance (Rti = G/(ω/ω0)α) and tissue elastance (Eti = H(ω/ω0)1-α). The normalization factor ω0 = 1 rad/sec is introduced in order to obtain meaningful units for the parameters G and H (3). The impedance of the airways (Zaw) in each pathway is given by: Zaw (ωn) = Raw + jωnIaw (Eq. 2) The Raw, Iaw and hysteresivity (14), defined as the ratio of η = G /H, of the tissue elements were the same in each pathway while the elastance (H) of the tissue elements followed a hyperbolic distribution between a minimum (Hmin) and a maximum (Hmax). The input impedance Z of the network is obtained as (20): Zin = FZaw ZLti ,min + Zaw ) F + ln( ZLti ,max + Zaw (Eq. 3) where F, ZLti,min and ZLti,max are given by F = ln(Hmax/Hmin), ZLti,min (ωn) = (η – j)Hmin/ωnα, and ZLti,max (ωn) = (η – j)Hmax/ωnα. By minimizing the root mean square difference between the model and the data (6) the five parameters (Raw, Iaw, η, Hmin, and Hmax) were determined. The mean H value (Hmean) was estimated as the expected value of the distribution function and was calculated from the estimates of Hmin and Hmax as: Hmean = H max − H min (Eq. 4) F The tissue damping, G, was calculated as G = ηHmean. Dynamic Nonlinearity of Respiratory Mechanics When applying a broad-band input, one way to probe system nonlinearity is to measure how the elastic and viscous moduli depend on the amplitude of the input. Another way of quantifying nonlinearity is via the so-called harmonic distortion index, which estimates the amount of both harmonic distortion and cross talk in the output signal resulting from system nonlinearities (42). For a broadband input, the coefficient of harmonic distortion Kd is defined as (54): Kd = P NI /PTOT x 100 (%) (Eq. 5) where PTOT is the total power in the output and PNI is the output power due to system nonlinearities only, i.e., the power at non-input frequencies. Since only nonlinearities and noise can produce output energy at non-input frequencies, the values of Kd were also corrected for nonzero energy at non-input frequencies (54). The advantage of using Kd is that it can be calculated from a single impedance measurement, whereas the traditional method of characterizing nonlinearity requires the measurement of the moduli at several distinct amplitudes. The Kd in a linear system is zero. In a nonlinear system driven by sinusoidals, the Kd measures the distortion and crosstalk due to system nonlinearities. Measurements of Mechanical Failure of Lung Tissues To assess the strength of the alveolar wall in the parenchyma, failure test of lung tissue strips were carried out. Following the measurements of respiratory function, an additional dose of pentobarbital (70 mg/kg) was injected intraperitoneally to each animal (n = 6 in each group), the thorax was opened, and the animals were exanguinated by severing the inferior vena cava. The heart, lungs, and trachea were carefully resected en bloc and rinsed in phosphate-buffered saline (PBS) (Sigma). The experimental set up was described previously (53). Parenchymal tissue strips having dimensions of 2.0 - 3.0 mm x 0.7 - 1.0 mm x 0.7 - 1.0 mm in length, width and thickness, respectively, were carefully prepared from each lung and the pleura were removed with the use of a razor. Each end of the tissue strip was fixed by cyanoacrylate glue to small metal plates attached to straight steel wires. The assembly was placed in a horizontal tissue bath filled with PBS at room temperature, with one wire attached to a computer-controlled lever arm containing a force transducer (Model 300B, Aurora, ON, Canada). We followed the method reported by Tanaka and Ludwig (47). Briefly, each strip was stretched at a rate of 0.2 mm/sec until the sample separated into two pieces. Due to the limitation of the maximum displacement of the level arm, the strips were first stretched to approximately 3 times their unstretched length before the recording of the displacement and force signals was started. The strain was defined as the total displacement in length normalized by the unstretched length of the samples. A transient decrease in the force (see arrows in Fig. 8) was defined as “the failure stress”, indicating that fibers in the alveolar walls started to break during stretching. Two or three strips were prepared and measured from each animal, and the average value of the failure stress was used for statistical analysis. Bronchoalveolar Lavage Whole lung lavages were performed with 1 ml of PBS twice via the tracheal cannula following the impedance and P-V curve measurements (n = 5 in each group). The return volume was measured, and the bronchoalveolar lavaged fluid (BALF) was centrifuged. The cell pellet was re-suspended in red blood cell lysis buffer (0.01% NH4Cl) and brought up to the initial lavaged return volume for total cell count by hemocytometry. Slides containing 500 - 1000 cells were prepared using a cytocentrifuge, and stained with rapid Wright’s stain. Differential cell analysis was performed by manual counting under a light microscope. Lung Histology and Morphometry After mediastinal dissection, the lungs were perfused with 10% buffered formalin via the tracheal cannula at an airway pressure of 25 cmH2O for at least 20 min (n = 5 in each group). The fixed lungs were embedded in paraffin, sectioned and stained with hematoxylin and eosin for histological analysis. We calculated the average distance between alveolar walls, the mean linear intercept (MLI), according to established methods (10) using a light microscope. For each pair of lungs, 10 histological fields were evaluated. BALF Elastase-like Activity Elastase-like activity in BALF was measured using the protocol by Dhami et al. (9). Briefly, BALF samples were lyophilized and reconstituted in water to make a fivefold concentrated solution. An assay buffer of 0.2 M Tris · HCl, pH 8.0, was prepared and then, 100 µl of assay buffer, 50 µl of substrate (0.5 mg/ml of N-succinylAla-Ala-Ala-p-nitroanilide), and 50 µl of BALF were added. All samples were assayed in duplicate. EDTA (10 mM) was added to the samples to inhibit metalloelastase (13). Negative control of 150 µl of assay buffer and 50 µl of substrate were used. We assessed background absorbance of each BALF sample by incubating 150 µl of assay buffer with 50 µl of each sample. This value was then subtracted from the absorbance of the test wells. The absorbance of the wells is measured at 405-nm wavelength using a spectro-photometer. Whole Lung Collagen Content Collagen content was assessed by measuring the hydroxyproline content of the tissue as previously described by Woessner (51). Lungs from the control (n = 7) and the PPE-treated (n = 7) mice were lyophilized for 12 h to dry weight, measured, minced. The lung sample was then hydrolyzed with 4 ml of 6 N HCl at 100°C for 6 h. One ml of the hydrolysate was then taken and evaporated. The powder was reconstituted with 1 ml of distilled H2O (dH2O) and re-evaporated. The powder was then reconstituted with 5 ml of dH2O. Hydroxyproline (Sigma) standard solutions of 0 - 10 µg/ml were prepared. Sample solution (2 ml) was taken and oxidized with 1 ml of Chloramine-T (Sigma) for 20 min. The reaction was then stopped with 1 ml of 3.15 M perchloric acid. After 5 min, 1 ml of p-dimethylaminobenzaldehyde solution was added. The sample was vortexed, incubated in a 60°C bath, and then cooled under tap water for 5 min. The absorbency of the solutions was determined at 557 nm using a spectro-photometer. The hydroxyproline concentration was determined from the standard curve. Whole Lung Elastin Levels The lyophilized was placed into 1 ml of 0.25 M oxalic acid. The suspension was then heated at 100°C for 1 h. The specimen was centrifuged, and the supernatant was collected. The above procedure was repeated for a total of five times until all the insoluble elastin had been converted into a soluble product (α-elastin) (7). One ml of the collected supernatants for each mouse was then dialyzed against water using 15,000 molecular-weight cutoff dialysis membrane (Spectrum, Huston, TX). The levels of α-elastin were then determined using Fastin-elastin assay (Biocolor, Belfast, N. Ireland) following the specific protocol outlined in the kit. Statistical Analysis All data were expressed as means ± standard deviation (SD). Student's t-test and repeated-measure two-way analysis of variance were used to evaluate the significance of differences between means and variances, with P < 0.05 as the level of significance. RESULTS Histopathology Fig. 1A and B shows representative alveolar structures of a control and a PPE-treated lung stained with hematoxylin and eosin three weeks following initial treatment. Significant enlargement of the alveolar air spaces was observed in lung samples from PPE-treated mice compared to control. Both the mean and the SD of the MLI in the PPE-treated group (83 ± 21 µm) were significantly larger than in the control group (43 ± 3 µm)(P = 0.001). There were no significant differences in body weight between PPE-treated (24.9 ± 0.9 g) and control groups (25.2 ± 1.1 g). BALF Analysis There were no significant differences in BALF total cell numbers between PPE-treated (3.4 ± 1.0 x 104 /ml) and control groups (3.8 ± 0.9 x 104 /ml) and over 95% of the cells were macrophages in both groups. No eosinophils and only 0.5% neutrophils were observed in both groups. Additionally, no elastase-like activity was detected in BALF of the control and PPE-treated mice, suggesting that direct elastolytic activity of PPE had already diminished at three weeks following treatment as reported previously (40). Pressure-volume Curves and Dynamic Respiratory Mechanics Fig. 2 compares the quasi-static P-V curves in the two groups obtained during volume-controlled inflation from end-expiratory volume. The pressure was significantly lower in the PPE-treated mice than in the control (P < 0.001). The average quasi-static elastance value, defined as a slope between 0 and 1.15 ml of inflated volume, was significantly lower in the PPE-treated mice (18.8 ± 2.8 cmH2O/ml) than in the control (26.5 ± 1.7 cmH2O/ml) (P < 0.001). Fig. 3 shows representative cases of the dynamic respiratory system resistance and elastance (calculated from the reactance where elastance = -2πf * reactance) as a function of frequency, and the fits of the mathematical model (20) to the data in representative control and PPE-treated mice at PEEP = 3 cmH2O. The measured data were fit well by the model at all PEEP levels in both groups. As a function of PEEP, all the values of tissue elastance (H) parameters, Hmin, Hmax and Hmean, were significantly PEEP-dependent (P < 0.001) and decreased with increasing PEEP in both groups (Fig. 4). The values of Hmin, Hmax, and Hmean were significantly lower in the PPE-treated group than in the control (P < 0.001)(Fig. 4A and B). The largest difference in Hmax between the control and the PPE-treated mice was at the highest (9 cmH2O) PEEP. Hysteresivity η was significantly PEEP-dependent in the PPE-treated mice (P < 0.001) but not in the control, and was significantly higher in the PPE-treated mice at PEEP ≥ 6 cmH2O (Fig. 5A). Airway resistance Raw was significantly PEEP-dependent (P < 0.001), but there was no significant difference between the groups (P = 0.78)(Fig. 5B). The tissue damping (G) was significantly PEEP-dependent (P < 0.001) and significantly lower in the PPE-treated mice (P < 0.001) (Fig. 5C). Dynamic Nonlinearity The harmonic distortion Kd, a measure of dynamic nonlinearity of the respiratory system, was significantly PEEP-dependent (P < 0.001) and decreased implying more linear behavior when PEEP was increased in both groups (Fig. 6). The Kd was significantly larger in the PPE-treated group than in the control (P < 0.001). Figure 7 shows the linear relationships between Hmin or Hmax and Kd including data from all PEEP levels. There were significant correlations between Hmin and Kd both in the PPE-treated (Kd = 0.51Hmin – 1.56)(P < 0.001, r = 0.83) and control groups (Kd = 0.36Hmin – 2.21) (P < 0.001, r = 0.90) (Fig. 7A). The slopes of the two lines were significantly different (P < 0.05). There were also significant correlations between Hmax and Kd both in the PPE-treated (Kd = 0.13Hmax – 2.44) (P < 0.001, r = 0.86) and the control mice (Kd = 0.14Hmax – 0.82)(P < 0.001, r = 0.86) (Fig. 7B). The two lines were nearly parallel and there was no significant difference in slopes between the two lines (P = 0.48) but the intercept of the PPE-treated group was relatively higher which nearly reached a statistical level (P = 0.07). There were also significant correlations between Hmean and Kd both in the PPE-treated (Kd = 0.29Hmean – 2.04) (P < 0.001, r = 0.93) and the control groups (Kd = 0.23Hmean – 3.00) (P < 0.001, r = 0.94), and the slopes of the two lines were significantly different (P < 0.05) (data not shown). Following the PPE treatment, each point moved to the left at an equivalent PEEP, implying stronger nonlinearity per unit tissue elastance in the PPE-treated group. Lung Tissue Failure Tests Fig. 8 shows representative traces of parenchymal tissue strip failure tests from a PPE-treated and a control mouse. The stress in each tissue strip increased with strain until a maximum was reached. Next, the stress started to drop in discrete steps suggesting that groups of alveolar walls broke within the tissue. Finally, the stress levels returned to zero when the samples separated into two pieces. Table 1 shows the summary of failure test results for all parenchymal tissue strips. The stress that developed just prior to the strips failing, defined as the failure stress, was significantly lower by 40% in the PPE-treated group than in the control (P = 0.002), demonstrating that the PPE-treated parenchymal tissues were weaker than control tissues. There were no significant differences in the strain values at failure (P = 0.56). Lung Hydroxyproline and Elastin Levels The levels of hydroxyproline and α-elastin are expressed as µg/mg dry weight of lung tissue (Table 2). There were no significant differences in dry lung weight between the control and the PPE-treated mice (P = 0.81). The levels of hydroxyproline, a measure of total collagen, were significantly higher in the PPE-treated mice by 48% than in the control (P = 0.002). The levels of α-elastin were lower by 13% in the PPE-treated mice, but the difference was not statistically significant (P = 0.16).