INTRODUCTION

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SMALL RODENTS HAVE BEEN WIDELY used to investigate the physiology and pathogenesis of pulmonary disease because large amounts of similar animals can be found, thus allowing the easy reproducibility of a variety of models of pulmonary disease. Rats and mice are easily raised and maintained, and pure-bred strains can be found. In addition, the whole lung can easily fit in one slide, thus yielding an overall morphological study.

Lung parenchymal strip has been used to study the behavior of pulmonary tissue in response to different contractile agonists and antagonists, as well as to examine the tensile and viscoelastic properties of the pulmonary parenchyma (3, 21). Additionally, some studies have indicated that the connective matrix structure may determine the mechanical behavior of lung tissue (3, 9, 17). However, interspecies differences have been reported (6, 22). These facts highlight the importance of understanding how tissue mechanical properties vary among different rodents to better describe a specific disease. Finally, little information about mice lung mechanics (6), as well as mice parenchymal strips properties, is presently available (18).

To determine the possible differences between mouse and rat lung parenchymal micromechanical behaviors and whether specific lung parenchymal characteristics could be related to any mechanical profile, we analyzed rat and mouse resistance (R), elastance (E), and hysteresivity () during sinusoidal oscillations of lung parenchymal strips and their corresponding histopathology. The collagen-elastin matrices of both species were also studied.

	MATERIALS AND METHODS

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Tissue preparation. Five male Wistar rats (250-300 g) and five male BALB/c mice (25-30 g), with age and maturation matched, were sedated (diazepam: 5 and 1 mg ip, respectively) and anesthetized with pentobarbital sodium (20 mg/kg ip). Then the animals were tracheotomized, and a snugly fitting cannula (1.4 and 0.8 mm ID, respectively) was introduced into the trachea. The lungs were removed en bloc at the functional residual capacity and rinsed in a modified Krebs-Henseleit (K-H) solution containing (in mM) 118.4 NaCl, 4.7 KCl, 2.5 CaCl₂ · H₂O, 0.6 MgSO₄ · H₂O, 1.2 K₃PO₄, 25.0 NaHCO₃, and 11.1 glucose, pH 7.40 and 6°C. A 3 × 3 × 10-mm strip of subpleural parenchyma was cut from the periphery of each left lung. Pleural tissue was removed, and the strips were kept in a recirculating bath of iced K-H solution that was continuously bubbled with a mixture of 95% O₂-5% CO₂.

Apparatus. Parenchymal strips were suspended vertically in a K-H organ bath maintained at 37°C and continuously bubbled with 95% O₂-5% CO₂. Metal clips made of 0.5-mm-thick music wire were glued to both ends of the tissue strip with cyanoacrylate. One clip was attached to a force transducer (FT03, Grass Instruments, Quincy, MA), whereas the other one was fastened to a vertical rod. This fiberglass stick was connected to the cone of a woofer, which was driven by the amplified sinusoidal signal of a waveform generator (3312A function generator, Hewlett Packard, Beaverton, OR). A sidearm of the rod was linked to a second force transducer (FT03, Grass Instruments) by means of a silver spring of known Young's modulus, thus allowing the measurement of displacement. Length and force output signals were conditioned, fed through eight-pole Bessel filters (902LPF Frequency Devices, Haverhill, MA), analog-to-digital converted (DT2801A, Data Translation, Marlboro, MA), and stored on a computer. All data were collected using LABDAT software (RHT-InfoData, Montreal, Quebec). The frequency response of the system was dynamically studied by using calibrated silver springs with different elastic Young's modulus. Neither amplitude dependence (<0.1% change in stiffness) nor phase changes with frequency were detected in the range from 0.01 to 14 Hz. The  of the system was independent of frequency and had a value <0.003.

Preconditioning. Cross-sectional, unstressed area (A₀) of the strip was determined from volume and unstressed length, according to A₀ = vol/L₀. Basal force (F_B) for a stress of 10 g/cm² was calculated as F_B (g) = 10 (g/cm²) · A₀ (cm²) and adjusted by vertical displacement of the force transducer (2, 16, 25). The displacement signal was then set to zero. Once basal force and displacement signals were adjusted, the length between bindings (L_B) was measured by means of a precision caliper. Instantaneous length during oscillation around L_B was determined by adding the value of L_B to the measured value of displacement at any time.

Measurements of parenchymal mechanics. To calculate tissue R, E, and , force-length curves were analyzed (9). Instantaneous average cross-sectional area (A_i) was determined as A_i = vol/L_i (cm²), where L_i is instantaneous length. Instantaneous stress (_i) was calculated by dividing force (F; g) by A_i (_i = F/A_i).

(1)

(2)

1

(3)

Morphometric analysis. At the end of the experiments, the organ bath was removed, and the parenchymal strips were frozen at the tension maintained during the experiment by rapid immersion in liquid nitrogen. Frozen strips were fixed in Carnoy's solution (ethanol-chloroform-acetic acid, 70:20:10) at 70°C for 24 h. Solutions with progressively increasing concentrations of ethanol at 20°C were then substituted for Carnoy's until 100% ethanol was reached. The tissue was maintained at 20°C for 4 h, warmed to 4°C for 12 h, and then allowed to reach and remain at room temperature for 2 h (17). After fixation, the tissue was embedded in paraffin. Four-micrometer-thick slices were obtained by means of a microtome. They were stained with hematoxylin-eosin.

Statistical analysis. The normality of the data (Kolmogorov-Smirnov test) and the homogeneity of variances (Levene median test with Lilliefor's correction) were tested. In all cases, both conditions were satisfied, and thus one-way ANOVA for repeated measurements was used to determine the effect of frequency on E, R, and  in each group. If multiple comparisons were required, Student-Newman-Keuls test was applied. Comparisons between E, R, , and morphological data between the two species were analyzed through t-test.

	RESULTS

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The effects of different frequencies on E, R, and  are shown in Fig. 1. In both species, R had a negative and E a positive dependence on frequency, whereas  remained unchanged.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Resistance (logarithmic scale), elastance, and hysteresivity in rats and mice at different frequencies (0.03, 0.1, 0.3, 1, and 3 Hz). Values are means ± SE of 5 animals in each group. a-e Different letters mean significantly different values at the various frequencies in the same group, P < 0.05. * Values significantly different between groups, P < 0.05.

Values of E were higher in mice than in rats (24.9%), whereas  was larger in rats (45.3%), as shown in Fig. 1. These differences were not frequency dependent. R was similar in both species (Fig. 1).

Table 1 shows the results of the histological analysis. Although the amount of BW was similar between the two species, mouse parenchymal strips had significantly more AW (P = 0.0008) and significantly less BVW (P = 0.004). Mouse lungs also had smaller L_m than rats (P = 0.001). The two species showed similar collagen and total elastic system fiber contents (Table 1), but mouse lung tissue had significantly more Oxy fibers and significantly less Ela and FDEF than that of the rat (Table 1).

No correlation could be established between mechanical and histological data.

	DISCUSSION

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This study shows that the oscillatory mechanical profiles of rat and mouse parenchymal strips differ. Although both species had similar R values, E values were higher and  was smaller in mice than in rats (Fig. 1). Rat and mouse lung tissue strips showed dependence of E and R on frequency, in agreement with previously reported data (2, 4, 22).

Before the results are discussed, technical issues warrant consideration. In the present study, strips could not be obtained from lungs of similarly sized animals, but the strips obtained had precisely the same size and were obtained from the same lung region. When tissue mechanics are analyzed, the morphological parameters of the parenchymal strip seem to be the major factors. In this way, the most important sample characteristic is the lung region from which the strip is obtained. The subpleural parenchymal strips are a sound model of parenchymal lung behavior, whereas in more central strips the amount of BW may play an important role in determining the hysteretic response (21). The use of instantaneous stress allows stress normalization when strips from different origins or species are compared.

Our results demonstrated similar R values in mice and rats, whereas tissue E was higher and  was smaller in mice than in rats (Fig. 1). Gomes et al. (6) also described a more rigid respiratory system in mice than in rats, suggesting that the differences in the relative compliances of lung and chest wall between species could be responsible for these differences because the increase in lung compliance with body weight has a greater power than that of the chest wall. Additionally, the authors suggested that the more rigid respiratory system could probably be the result of a relatively smaller air space volume in mice. It should be stressed that our study highlights the specific role of lung tissue E, because the model used is completely independent of the chest wall properties. Additionally, our results also showed smaller L_m in mice than in rats.

We found that  varied between rats and mice. In another study comparing mechanical properties among different species, Gomes et al. (6) reported that  should be independent of animal size, because tissue damping and E had similar power variations with body weight, thus resulting in a constant ratio. However, the comparison between their results and ours is unwarranted, because the methods as well as the assumptions are different, and their results pertain to the respiratory system, whereas we are dealing with isolated lung strips.

Interestingly, taking into consideration tissue strips from various species oscillated at the same frequency and tension as ours (1, 3, 13, 22),  varies from 0.075 (1) to 0.121 (3), E ranges between 2.0 (22) and 7.4 × 10⁴ N/m² (1), and R varies from 0.1 (13) to 3 × 10² N · s · m² (22). These variations are remarkably smaller than those presented by mechanical parameters gathered from the whole lung: pulmonary R ranges between 0.98 (10) and 23 cmH₂O · l¹ · s (19), and dynamic E varies from 9.6 (10) to 336 (19) cmH₂O/l.

Although a statistical correlation between mechanical and histological data could not be established, our mechanical results were accompanied by a greater volume proportion of AW and smaller L_m in mice than rats (Table 1). On the other hand, the anatomic makeup has a close correlation with the location from which the strip is cut from the lung, i.e., obtained from lung periphery or central regions, an important issue when constriction models are studied (17).

When different species are compared, with strips cut from the lung periphery, the volume proportion of AW can also represent different alveolar sizes among animals. In our work, this hypothesis was supported by L_m values. Salerno et al. (22) reported different anatomic composition, i.e., the relative amounts of BW, BVW, and AW, when comparing rat and guinea pig parenchymal strips. These authors suggested that the volume proportion of the different anatomic structures comprising the parenchyma could play a role in the transmission of stress to the airway wall via parenchymal attachments. Haber et al. (8) showed that tissue elastic properties do not normally determine lung distensibility. They observed that it appears more likely that changes in tissue distensibility act through changes in the size of air spaces and that alteration in the density of elastic fibers may influence the distensibility to the extent that there is an accompanying change in the size of air spaces, which implicates alveolar size as the major determinant of pulmonary E (8). The higher E found in mice than in rats could probably be related to the smaller L_m value in mice. These results are in agreement with previous suggestions (6). As our model is independent of the chest wall properties, it strengthens the possible role of the pulmonary anatomical composition in determining distensibility.

Different tissue mechanical properties between rats and mice were also accompanied by diverse extracellular matrix composition. Mice showed a higher amount of Oxy and a smaller content of elastin and FDEF than did rats (Table 1). Although some authors (16) suggest that  does not depend on the amount of tissue participating in the expansion, our previous study on the extracellular matrix in silicotic mice (2) showed that  was dependent on ELA + FDEF. There is no report on the relative expression of each component of the elastic system between rats and mice. The elastic system is composed of Oxy, Ela, and FDEF, defined according to increasing amounts of elastin and fibril orientation (15). The elastic properties of each fiber depend on their amorphous component, i.e., elastin (15, 20). Thus the Oxy fibers, composed solely of microfibrils without elastin, do not elongate under mechanical stress. These fibers would prevent tissue overstretching. The Ela fibers, which contain both microfibril bundles and a small content of amorphous material, are expected to display elastic properties that are intermediate between those of elastic and Oxy fibers (15). Some authors also demonstrated an important relationship between elastin and collagen in the stress-strain behavior of arteries, suggesting that an interweaving of collagen and elastic fibers permits the elastic fibers to sustain a higher stress (20). Thus the higher content of Oxy fibers and the smaller amount of the other elastic fibers could probably lead to the stiffer lung found in mice. The differences in the mechanical properties probably cannot be attributed to collagen content, because it was identical in both species.

In this way, the distinct behavior of rat and mouse  found in the present work could be dependent on the differences in extracellular matrix composition between both species. Little is known about the mechanisms that might govern the rheology of intraparenchymal connective tissues and fiber networks. Dissipation in connective tissues may originate at the microstructural level, but elasticity and  of the connective tissue network would appear to be more a property of the fiber matrix than of the material from which the fiber is constituted (4). The energy dissipation in such systems may be governed 1) by contact phenomena between stress-bearing connective tissue fibers, which could occur at the molecular level within elastin or collagen fibers, 2) by shearing of the glycosaminoglycan ground substance between fibers, or 3) at surfaces of direct fiber-fiber sliding contact (4, 13, 14, 25).

In conclusion, we showed that mouse and rat parenchymal strips have diverse mechanical behaviors. The differences in tissue mechanics between both species were accompanied by different anatomical makeup and extracellular matrix composition. The results evidence a diverse proportion of specific components of the elastic system between mice and rats, which should be considered when particular diseased states are modeled.