Difference Between C57bl 6 And Balb C

[Queila Neubecker]

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C57BL/6 and BALB/c mice are the most widely employed mouse strains in experimental lung research, in particular for modeling human respiratory diseases1,2. Several respiratory diseases such as interstitial lung disease (ILD) and chronic obstructive pulmonary disease (COPD) are characterized by a change in lung elastance3,4,5,6. It is also well known that aberrant lung elastance alters lung function7. For example, both ILD and COPD, characterized with higher and lower than normal elastance, respectively, lead to a decline in the spirometric measurement of the forced expiratory volume in 1 s (FEV1).

Whether C57BL/6 mice have a greater lung elastance than BALB/c mice because of a smaller lung volume, a stiffer lung tissue, or a combination thereof, is unknown. Untangling the determinants of this conspicuous strain difference may hint investigators on the most suitable mouse strains to choose from for addressing key questions in specific models of human respiratory diseases. This study was undertaken to determine whether a smaller lung volume or a stiffer lung tissue accounts for the greater lung elastance of C57BL/6 than BALB/c mice.

Protocol to measure lung tissue mechanics of the right inferior lobe in vitro. Each lobe was subjected to 3 consecutive sequences of sinusoidal small-amplitude strain oscillations for 2 min followed by a half-sine stretch of 30%. A representative strain trace is shown in (A). The green section prior to the second and the third 30% stretches are locations where the mechanical properties of the lobe (i.e., elastance, resistance and hysteresivity) were calculated during the small-amplitude oscillations. They were selected to be away (i.e., 1 min) from the large 30% stretch, where the mechanical properties had nearly reached a steady-state. The blue and red sections are locations of the second and third half-sine stretches of 30%, where the mechanical properties of the lobe were calculated during the large-amplitude oscillations. They were selected because they were the first 30% stretches where the mechanical properties of the lobe had reached a near steady-state. The corresponding force trace before, during, and after the second 30% stretch is shown in (B). The corresponding force-strain trace is shown in (C). The blue and red lines are actual data fits during the 30% stretch and retraction, respectively. Together, they were used to calculate hysteresis (i.e., the area within the loop), which was then used to sequentially calculate resistance, hysteresivity and elastance. The traces in (A) and (B) look continuous but are, in fact, constituted of discrete data points sampled at 100 Hz, as can be seen in (C).

This study demonstrated that the lung size, measured by weight and volume displacement of a liquid by the excised lung, are similar between C57BL/6 and BALB/c mice. It also confirmed that the elastance of the respiratory system is greater in C57BL/6 than BALB/c mice when measured in vivo, as testified by increases in respiratory system elastance (Ers) and tissue elastance (H), as well as by decreases in the parameter K of Salazar-Knowles equation, lung compliance (C) and the volume at 10 cmH2O expressed in percentage of TLC (V10_TLC). Most volumes measured in vivo were also different between the two mouse strains, being smaller in C57BL/6 than BALB/c mice for TLC, VC, FRC and ERV, and inversely greater in C57BL/6 than BALB/c for RV. Finally, in vitro experiments on isolated lobes demonstrated that tissue elastance was greater in C57BL/6 than BALB/c mice, which was associated with a greater content of hydroxyproline. It is concluded that the lung elastance of C57BL/6 is greater than BALB/c mice mainly because of a stiffer lung tissue due, at least partially, to a greater content of collagen.

Several strains of mice are used to study lung mechanics and for modeling human respiratory diseases. Yet, C57BL/6 and BALB/c mice are the most widely employed1,2,9. These two strains exhibit different susceptibilities for the development of specific pathogenic traits reminiscent of human respiratory diseases in response to offending triggers. For example, while C57BL/6 are more prone than BALB/c mice for the development of pulmonary fibrosis upon exposure to bleomycin in models of idiopathic pulmonary fibrosis2,16, BALB/c are more prone than C57BL/6 mice for the development of methacholine hyperresponsiveness upon exposure to allergen exposure in models of asthma9,17 and for the development of emphysema upon exposure to cigarette smoke or elastase in models of COPD18,19. Understanding inherent differences in the mechanical properties of the lung between these two mouse strains may help in interpreting these varying strain susceptibilities. In turn, this may help for guiding the choice of strains in models of human respiratory diseases.

One striking lung difference between C57BL/6 and BALB/c mice is elastance. The elastance of the lung and the respiratory system is greater in C57BL/6 than BALB/c mice8,9,10,11,12,13,14. This is likely to confer either protection against or vulnerability for the development of specific pathogenic traits. Either way, inferring on the contribution of varying lung elastance to any specific pathogenic trait depends on whether this is due to a smaller lung or a stiffer lung tissue, which has never been delineated. Herein, a comprehensive characterization of lung mechanics was undertaken to determine whether this between-strain difference in lung elastance was due to a different lung volume or to a different stiffness of the lung tissue.

Six readouts were measured to compare respiratory system elastance in vivo between C57BL/6 and BALB/c mice. Importantly, these six readouts are not independent from each other. They are measured using different procedures, but are often sensitive to the same or similar underlying features. Four of them, namely Ers, H, Est and C, are sensitive to both lung volume and tissue stiffness. Ers and H, both indicators of elastance, were higher in C57BL/6 versus BALB/c mice. Concordantly, C, an indicator of respiratory system compliance, was lower in C57BL/6 versus BALB/c mice. Est was also numerically (but not statistically) higher in C57BL/6 than BALB/c mice. Together, these readouts confirmed that the elastance of the respiratory system is greater in C57BL/6 than BALB/c mice. The two additional in vivo readouts include K and V10_TLC. They are both purportedly insensitive to lung volume20,21, and thus exclusively sensitive to lung tissue stiffness. They are currently considered volume-independent indicators of the compliance of the respiratory system20,21. They were both significantly lower in C57BL/6 than BALB/c mice. This was the first hint suggesting that the lung elastance was greater in C57BL/6 than BALB/c mice because of a stiffer lung tissue.

Notably, all in vivo measurements in the present study, including lung volumes, were performed with an intact chest wall. Consequently, the differences in respiratory system elastance between C57BL/6 and BALB/c mice, reported herein and elsewhere8,9,10,11,12,14, may also be attributed to between-strain differences in chest wall mechanics. Although possible, Swedin et al.13 have demonstrated that lung elastance was also greater in C57BL/6 than BALB/c mice by measuring mechanics in open-chest conditions. This unequivocally confirmed that a greater lung elastance contributes, at least partially and perhaps totally, to the greater respiratory system elastance reported herein and by other8,9,10,11,12,14.

In the present study, further in vitro experiments were undertaken to exclude the confounding effect of the chest wall. The techniques employed also exclude confounding effects of all other in vivo factors on lung tissue mechanics, such as circulating or vagally-derived mediators affecting the level of airway smooth muscle activation20.

Firstly, the volume of the whole excised lung was measured in vitro by plunging it into Krebs and measuring the displaced liquid volume. The lack of difference between mouse strains confirmed that the lung volume at zero transpulmonary pressure is not different between C57BL/6 and BALB/c mice. This was also consistent with the lack of difference in the lung wet weight between the two mouse strains, as well as the lack of difference in their total body weight.

Secondly, lung tissue mechanics was investigated in vitro on an isolated lobe immerged in Krebs solution. This technique is useful because it directly assesses the mechanics of the lung tissue. The data demonstrated that tissue elastance was greater in C57BL/6 than BALB/c mice, confirming that the lung tissue is stiffer in the former than the latter.

The claim that the lung elastance of C57BL/6 is greater than BALB/c mice because of a stiffer lung tissue and not because of a different lung volume may sound conflicting given that most lung volumes (TLC, VC, FRC, ERV and RV) were significantly different between the two mouse strains. The results could have indeed been misleading if only a high lung volume (e.g., TLC) had been measured without measuring RV and without a complementary set of in vitro data. However, the lack of difference in weight and volume of the excised lung confirmed that the lung size of C57BL/6 and BALB/c mice is similar. The measurement of tissue mechanics on the isolated lobe in vitro also directly demonstrated that the lung tissue of C57BL/6 is stiffer than BALB/c mice. It is with these latter measurements, combined with the measurement of two volume-independent indicators of respiratory system compliance (K and V10_TLC), that it became clear that the bidirectional difference in volumes observed in vivo (i.e., lower in C57BL/6 than BALB/c mice when measured at positive pressures, while higher in C57BL/6 than BALB/c mice when measured at a negative pressure) were also driven by a difference in lung tissue stiffness. It is, of course, logical that a lung with a stiffer tissue should not only be harder to inflate above zero transpulmonary pressure, but also harder to deflate below zero transpulmonary pressure.

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