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1Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Subiaco, Western Australia; 2Department of Medical Informatics and Engineering, University of Szeged, Szeged, Hungary; and 3Department of Intensive Care and Neonatology, University Children's Hospital, Zurich, Switzerland
Submitted 26 February 2008 ; accepted in final form 11 June 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 h of mechanical ventilation to return to the original state. We propose a number of possible mechanisms for these observations and suggest that they are most likely explained by the unfolding of alveolar septa and the subsequent redistribution of the fluid lining the alveoli at high transrespiratory pressure. mouse; pressure-volume curve; total lung capacity; forced oscillation technique; lung softening
20 cm H2O transrespiratory pressure (Prs), lung volume continues to increase rather than reaching a plateau, which results in a double sigmoidal pattern in the PV curve and no definable TLC (15). This type of PV curve was noted in other mammalian species including the sea otter and fruit bat in the 1970s (9). However, despite knowledge of the existence of this phenomenon for some decades, little attention has been paid to the mechanisms responsible for the biphasic nature of the PV curves in these mammals. This study aimed to explore the mechanisms responsible for the double sigmoid PV curve observed in mice. We systematically examined the changes in lung mechanics associated with inflation over 20 cm H2O. The effect of derecruitment of lung units by degassing or forcing lung volume below functional residual capacity (FRC) on the changes in lung mechanics following inflation beyond a Prs of 20 cm H2O was also examined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Eight-week-old female BALB/c mice were purchased from the Animal Resource Centre (ARC; Murdoch, Western Australia) and housed in specific pathogen-free conditions with a 12-h:12-h light/dark cycle. All experiments were conducted with the approval of the Telethon Institute for Child Health Research Animal Ethics Committee and conformed to the guidelines of the National Health and Medical Research Council of Australia.
Animal Preparation
Mice were anesthetized with an intraperitoneal injection of a solution containing 40 mg/ml of ketamine (Troy Laboratories, New South Wales, Australia) and 2 mg/ml of xylazine (Troy Laboratories) at a dose of 0.1 ml/10 g body wt. Two-thirds of the dose was given initially to induce a surgical level of anesthesia. Once anesthetized, the mouse was tracheostomized and a 10-mm length polyethylene tubing (1.26 mm outer diameter: 0.86 mm inner diameter) was inserted into the trachea. The tracheal cannula was secured with surgical silk, and the mouse was placed inside a whole body plethysmograph and connected to a computer-controlled small animal ventilator (flexiVent; SCIREQ, Montreal, Quebec, Canada). The remaining third of the anesthetic dose was given, and the mouse was ventilated at 450 b/min with a tidal volume of 8 ml/kg and 2 cm H2O of positive-end expiratory pressure (PEEP). This ventilation setting allowed for the measurement of lung mechanics (see details below) without the need for paralysis.
Thoracic Gas Volume
Thoracic gas volume (TGV) was measured as described previously (7). Briefly, the trachea was occluded at end expiration (Prs = 0 cm H2O), and inspiratory efforts were induced by stimulation of the intercostal muscles with intramuscular electrodes. Six pulses of 2–3 ms in duration at 20 V were delivered over a 6-s period while recording changes in tracheal pressure (Ptr) and plethysmograph box pressure (Pb). TGV was calculated by applying Boyle's law to the relationship between Ptr and Pb after correcting for the box impedance (7).
Lung Mechanics
The volume dependence of lung mechanics was assessed during a slow (40 s) inflation-deflation (ID) maneuver between 0 and 20 cm H2O Prs. Inspiration was induced by applying a controlled negative pressure to the plethysmograph, and expiration was achieved by the slow equilibration of the plethysmograph to atmospheric pressure through a resistor. During the maneuver, an oscillatory signal was applied to the lung with a loudspeaker in-box setup via a wave tube, with the respiratory input impedance of the mouse measured as a load impedance on the wave tube as described previously (4). Briefly, the oscillatory signal contained 9 noninteger-multiple frequencies ranging from 4 to 38 Hz. The respiratory system impedance spectrum was calculated for these frequencies with 0.5-s data segments throughout the ID maneuver. The four-parameter model with a constant-phase tissue impedance (5) was fitted to the data obtained for each 0.5-s data segment to allow calculation of airway resistance (Raw) and inertance (Iaw) and coefficients of tissue damping (G) and elastance (H). The resistance and inertance of the tracheal cannula were subtracted from Raw and Iaw, respectively. The values of the small and physiologically insignificant Iaw are not reported. This system allowed simultaneous calculation of the corresponding PV curves, whereby pressure changes were tracked and the commensurate volume measurements were calculated by integrating the wave tube flow during the maneuver. Starting TGV was calculated before all ID maneuvers.
Experimental Protocol
Effect of inflation to Prs = 40 cm H2O.
Mice were prepared as described above (n = 4). A series of ID maneuvers were performed as follows; 0-20-0 (ID20), 0-40-0 (ID40), and 0-20-0 cm H2O (ID''20). Each maneuver was separated by 5 min of regular ventilation. PV curves were constructed for each of the IDs, and respiratory mechanics were tracked throughout.
Exploring a critical maximum Prs. Serial ID maneuvers were performed (n = 4) with increasing peak Prs as follows; 0-15-0, 0-20-0, 0-25-0, 0-30-0, 0-40-0, 0-20-0 cm H2O.
Response of lung mechanics after inflation to Prs = 40 cm H2O to degassing or negative transrespiratory pressure. A series of IDs were performed per Effect of inflation to Prs = 40 cm H2O. The mice were then either degassed (n = 3) or exposed to negative Prs (n = 3) (to force lung volume down to levels approaching residual volume) before a third inflation to 20 cm H2O. Partial degassing of the lungs was achieved by ventilating the mouse with 100% O2 for 10 min and occluding the tracheal cannula for 1 min. A Prs of approximately –7 cm H2O was achieved by slowly injecting 2 ml of air into the plethysmograph over a 2-s period. The positive plethysmograph pressure was held for 4 s before inflating the mouse to 20 cm H2O and allowing passive deflation per a regular ID maneuver. After a short period of regular ventilation, the mouse was then subject to a regular ID maneuver to 20 cm H2O (ID''20).
Time taken to reverse the effect of inflation to Prs = 40 cm H2O. In another group of mice (n = 5), an ID20, ID40, and ID''20 series of maneuvers were conducted as described above. This series was then followed by ID maneuvers from 0-20-0 cm H2O after 30 and 60 min of regular ventilation. In additional groups of mice (n = 5 in each), a series of 0-20-0 cm H2O or 0-40-0 cm H2O were performed with measurements of TGV and lung mechanics taken at baseline, after 30 min, and after 60 min of ventilation.
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RESULTS |
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PV curves. The PV curve for the initial inflation to 20 cm H2O (ID20) had the shape of a classical PV curve with an apparent plateau in volume as the Prs approached 20 cm H2O (Fig. 1). For the subsequent inflation to 40 cm H2O (ID40), the slope of the PV curve increased again beyond 20 cm H2O such that the PV curve developed a double sigmoidal pattern. Following this maneuver, inflation to 20 cm H2O (ID''20) demonstrated a significant elevation in the slope of the PV curve with a significant increase in the volume reached at 20 cm H2O (TGV20) [ID''20, 1.07 ml (SD 0.07) vs. ID20, 0.74 ml (SD 0.08); P = 0.001] but not in the starting lung volume (TGV0) [ID''20, 0.32 ml (SD 0.03) vs. ID20, 0.28 ml (SD 0.06); P = 0.23] compared with the ID20.
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Qualitatively, the pattern seen in the volume dependence of tissue damping (G), was similar to that observed in H. When Prs was increased beyond 20 cm H2O, there was little change in the viscous properties of the lung as volume increased. As with H, there was a significant decrease in G0 [ID''20, 6.2 cm H2O/ml (SD 0.6) vs. ID20, 10.2 cm H2O/ml (SD 1.0); P = 0.004], Gmin [ID''20, 5.2 cm H2O/ml (SD 0.1) vs. ID20, 8.8 cm H2O/ml (SD 1.2); P = 0.01], and G20 [ID''20, 15.9 cm H2O/ml (SD 1.4) vs. ID20, 21.5 cm H2O/ml (SD 1.8); P = 0.001] for the ID''20 compared with the ID20. These "shifts" in the volume dependence of G and H during the ID''20 maneuver, compared with the ID20, were mirrored when they were plotted against pressure (Fig. 2). Similarly, the pressure dependence, particularly of H, clearly changed trajectory when Prs reached 20 cm H2O during the ID40.
Critical inflation pressure. Average PV curves for the inflations to 15 and 20 cm H2O had the shape of typical PV curves. When inflation continued beyond 20 cm H2O to 25 cm H2O, there was a shift in trajectory of the PV curve in subsequent maneuvers (Fig. 3). Accordingly, the inflation limbs of the 0–30 cm H2O and 0–40 cm H2O maneuvers departed from the common paths of the initial inflations. The TGV at Prs = 20 cm H2O (TGV20) was significantly higher in the 0–20 cm H2O inflation following inflation to 40 cm H2O (ID''20) [ID''20, 1.14 ml (SD 0.13) vs. ID20, 0.78 ml (SD 0.17); P = 0.01], whereas the starting volume (TGV0) was not significantly elevated [ID''20, 0.40 ml (SD 0.07) vs. ID20, 0.35 ml (SD 0.16); P = 0.39] (Fig. 3).
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PV curves. The change in the standard PV curve following inflation to 40 cm H2O was as seen in the above experiments with a significant increase in TGV20 in the ID''20 compared with the ID20 for both groups of mice (Fig. 4). Partial degassing of the lung, following an inflation to 40 cm H2O, reversed the increase in TGV20 observed in the ID''20 PV curve [ID20 postdegas vs. ID''20, 0.76 ml (SD 0.14); 0.82 ml (SD 0.12); P = 0.59] and did not cause any change in TGV0 (P = 0.12). Applying a negative Prs decreased TGV20; however, TGV20 was still significantly higher than the TGV20 for the ID20 maneuver [ID20 postnegative Prs vs. ID''20, 0.76 ml (SD 0.11); 0.88 ml (SD 0.16); P = 0.04].
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PV curves. For this series, the TGV20 was elevated significantly in the ID''20 compared with the ID20 (Fig. 6). After 30 min of ventilation, TGV20 had decreased but was still significantly higher than TGV20 for the ID20 [ID''20 + 30 min, 0.94 ml (SD 0.09) vs. ID20, 0.81 ml (SD 0.06); P = 0.005]. After 60 min of regular ventilation, TGV20 had returned to baseline levels [ID''20 + 60 min, 0.83 ml (SD 0.06); P = 0.51] (Fig. 6).
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Repeated Inflations to 40 cm H2O
PV curves.
The first part of the PV curve (up to 30 cm H2O) for the ID40' + 30 min was higher than the initial PV curve obtained from the ID40. Above this point the curves coincided. The PV curve for the inflation to 40 cm H2O 60 min after the initial inflation coincided almost perfectly with the PV curve from the ID40' + 30 min (Fig. 7).
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Repeated Inflations to 20 cm H2O
PV curves. There was no difference between TGV0 after 30 [ID''20 + 30 min, 0.20 ml (SD 0.02)] or 60 min [ID''20 + 60 min, 0.19 ml (SD 0.02)] of ventilation compared with baseline [ID20, 0.23 ml (SD 0.07)] (P = 0.21) (Fig. 8). However, after 30 min of ventilation, TGV20 [ID''20 + 30 min, 0.67 ml (SD 0.04); P = 0.003] had decreased below baseline levels [ID20, 0.76 ml (SD 0.08)] and even further after 60 min [ID''20 + 60 min, 0.61 ml (SD 0.08); P = 0.013] of ventilation (Fig. 8).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the absence of being able to measure starting lung volume to construct PV curves, previous studies designed to examine this phenomenon have involved complete degassing of the lungs before inflation (15). Consequently, large pressures were required to reopen the lungs (>20 cm H2O) for the first inflation maneuver. Since such a maneuver is not used in studies where lung volume history is standardized, it is unclear whether inflation to these pressures has an impact on subsequent lung function measurements. In the present study, we were able to measure TGV, which allowed construction of absolute PV curves without prior degassing of the lung. Serial ID maneuvers demonstrated a classic sigmoidal PV curve when the peak pressure applied to the lung was 20 cm H2O or below. However, once 20 cm H2O was exceeded, the horizontal asymptote in the PV curve was no longer apparent with a second knee appearing in the PV curve, as shown previously (9, 15). As the maximum Prs was increased, the volume excursion increased with no apparent limit to total lung volume. The deflation limbs of these PV curves ran parallel to each other, resulting in increased hysteresis in the PV curve with increasing maximum pressure.
Our study has clearly shown that a Prs of about 20 cm H2O represents a "critical point" in the inflation of the mouse lung whereby something fundamental changes in the processes or structures that are involved. This observation may have implications for studies in mice where lung volume history is standardized. Evidence for the changes that occur at 20 cm H2O arises from a number of results including the significant decrease in baseline tissue elastance we observed. In the absence of a significant change in TGV at 0 cm H2O Prs, this suggested that the lung parenchyma was more compliant after this maneuver. As a result, a larger volume of air was inhaled for the same pressure excursion in subsequent maneuvers. It was also clear that this phenomenon could be largely attributed to the response of the lung parenchyma. Although there was some hysteresis in the volume dependence of Raw in the ID maneuver, this can be explained by the fact that a much lower Prs was acting on the airways for the equivalent TGV during deflation compared with inflation. There was evidence from our data that something fundamental was changing in the way the lung elastance responded to volume excursion at pressures beyond 20 cm H2O with a sharp change in the trajectory of the H vs. volume curves. At Prs approaching 20 cm H2O, there was a relatively constant increase in H, which we have described previously and which may be attributed partly to the progressive stiffening of the lung (13, 4, 1). However, beyond 20 cm H2O H and G stopped increasing abruptly and remained relatively constant with no change in dynamic elasticity or viscosity of the lung despite a relatively large change in lung volume.
We investigated three separate strategies for "recovering" the changes in lung mechanics observed following inflation above 20 cm H2O: 1) partially degassing the lung, 2) applying a negative transrespiratory pressure such that lung volume fell below FRC, and 3) allowing spontaneous recovery. All of these strategies, to varying degrees, were able to recover the shift in the PV curve and volume dependence of tissue elastance. The most effective method was degassing the lung, which resulted in recovery of all the characteristic points on the H vs. TGV curve back to baseline levels. Although a negative transrespiratory pressure maneuver was not able to recover the H vs. TGV curve and V20 completely, all of these parameters had shifted in the direction of the original (ID20) values. Similarly, after 60 min of regular ventilation, although not all parameters had returned to baseline levels, given enough time, the changes in lung mechanics observed following inflation to 40 cm H2O spontaneously revert to baseline conditions.
There are a number of possible explanations for the appearance of a second knee in the mouse PV curve. First, one obvious explanation could be the contribution of the chest wall since we measured Prs rather than transpulmonary pressure throughout this study. However, studies in open-chested mice (Z. Hantos, unpublished observations) have demonstrated that the second knee is still present, which suggests that the contribution of the chest wall is unlikely to explain this phenomenon. Alternatively, the double sigmoid PV curve may be a result of the appearance of a "second" lung. By this we mean a portion of the lung that is not recruited until the critical pressure (20 cm H2O) is reached. This could be in the form of collapsed lung units that are not recruited until 20 cm H2O (recruitment of atelectic regions of the lung) or a second population of alveoli, a possibility raised by Soutiere and Mitzner (15), that are present but do not expand until this pressure is reached. These possibilities are consistent with the observations about the behavior of lung mechanics in this study following inflation to 40 cm H2O. The upward shift in the PV curve and the widening of the H vs. TGV curve, if this explanation is true, could be a result of the stability of these lung units/alveoli once they have been opened by inflation above 20 cm H2O. Upon subsequent inflations they then open uniformly with the other regions of the lung that are usually inflated when Prs is below 20 cm H2O. As a result, the stable part of the H vs. TGV curve, which is roughly associated with the central part of the PV curve where the bulk of the volume excursion occurs, is stretched because the previously opened lung units inflate smoothly during subsequent inflations, resulting in a more compliant lung and a larger volume of inhaled air.
This hypothesis is also consistent with the response to the recovery maneuvers. By degassing the lungs or forcing the lung below FRC, the reacquired lung units/alveoli would completely collapse, thus resetting the system to its original condition with 20 cm H2O needed to reopen these "lost" units. However, this explanation also requires that mice have an unused lung reserve that is not accessible under baseline conditions unless Prs exceeds 20 cm H2O. This seems like an unusual biological system, and one would have to wonder under what circumstance a mouse would be required to access this reserve. If there were a second population of alveoli or previously atelectic regions of the lung were being reopened, one would expect morphological evidence of an increase in alveolar number or evidence of a new population of alveoli (subset with a smaller size than the others) at pressures beyond 20 cm H2O. A very recent study by Namati and colleagues (10) using confocal imaging of isolated lung preparations found that, at transrespiratory pressures above 20 cm H2O, the alveoli appeared to become smaller and more numerous, suggesting that a "secondary" population of alveoli is recruited at high Prs.
Critical to this argument is what Prs a 20-g mouse is capable of generating. Equations for allometric scaling of TLC for mammals (16) would suggest that the TLC for a mammal of this size should be 0.85 ml, which is similar to the TGV20 observed in our study for the ID20 maneuver and suggests that, by inflating beyond this pressure, we may be inducing a response that is a result of a process beyond the normal operating range for a mouse lung in vivo. If we are operating in a pressure range that may not be physiological for a mouse, then it is entirely possible that we are simply exceeding the structural limit of the lung and it is possible to inflate mice to this extent due to their highly compliant chest wall (8). There is some data reporting transpulmonary pressures up to 30 cm H2O in spontaneously breathing mice in response to inhaled irritants (17) although these pressures only seemed to result in volume excursions of 0.2 ml; data from Leith's studies from the 1970s (9) suggest that small animals are quite capable of producing large transrespiratory pressure with a fruit bat producing a spontaneous breath with a Prs up to 60 cm H2O. If 20 cm H2O did represent a critical pressure limit, then one would predict that applying a pressure twice this amount would result in severe structural damage that would alter the mechanical properties of the lung. However, we had no evidence of irreversible damage to the system in our data with simple maneuvers, such as degassing the lung and recovering the lung to its baseline condition from a lung mechanics point of view. There was also a high level of consistency in the repeated inflations to a Prs of 40 cm H2O. Similarly, previous studies that have fixed the lung at Prs values up to 60 cm H2O showed no evidence of gross structural damage upon examination of histology (15).
An alternative explanation for this phenomenon can be found by considering surface tension at the alveolar surface. A large portion of the mechanical properties of the lung can be explained by the surface tension at the air liquid interface (2). As a result, it is entirely possible that by inflating the mice to such high lung volumes we are altering the surface acting forces in the lung in such a way as to alter the compliance of the lung. This could occur in a number of ways, either by stimulating the release of additional surfactant by mechanical stretch of alveolar type II cells (19, 11, 3) or by other physical mechanisms, such as redistribution of the available surfactant pool or a change in the surface tension of the air-liquid interface, resulting from thinning of this layer at high lung volumes (20). There is some evidence to support this mechanism as a potential explanation in other murine species. Nicolas et al. (12) have shown that exercising rats will increase their tidal volume by up to 300%, resulting in a significant increase in their surfactant pool. It was proposed that a large portion of this increased surfactant pool originated from direct stimulation of alveolar type II cells. This hypothesis is consistent with the primary observations in this study in that changes in surface acting forces could increase the compliance of the lung, resulting in increased TGV20 and decreased tissue elastance. The recovery of these parameters by degassing and negative transrespiratory pressure requires that the reverse process occurs during these maneuvers such that compliance is decreased back to baseline levels. Similarly, the spontaneous recovery of lung mechanics back to baseline levels could be attributed to the progressive uptake of surfactant. However, although this hypothesis provides an explanation for the effect of inflation above a Prs of 20 cm H2O on the subsequent changes in lung mechanics, it does not provide an explanation for the appearance of the second sigmoid in the PV curve, which is more consistent with a recruitment phenomenon.
As suggested above, it is possible that this inflection point in the PV curve is a result of the recruitment of a second population of alveoli by way of a mother/daughter alveolar structure, whereby the daughter (secondary) population of alveoli are recruited at high pressure via the pores of Kohn (10). However, this assertion is based on data using techniques that are only able to visualize the peripheral surface of the lung, which may not be able to take into account changes in alveolar shape and, as such, may not be representative of the changes occurring in the lung as a whole. Another alternative is that, rather than recruiting additional alveolar "units," the recruitment that occurs at the start of the second sigmoid in the mouse PV curve is a result of a change in the configuration of the existing alveoli. The configuration of each individual alveolus, once they begin to inflate, changes considerably during a single ID event. The change in configuration of the septa in an individual alveolus is dependent on the counteracting forces from the tethering of the septa to adjacent alveoli and the surface tension as a result of the fluid lining the alveolar surface. The interplay between these forces, in conjunction with the irregular shape of the alveolus, is thought to cause the septal folding observed in corners of the alveoli at moderate distension pressures (18). In light of this observation, one potential explanation for the appearance of the second sigmoid in the mouse PV curve is that 20 cm H2O is the point where these regions begin to unfold. This hypothesis is consistent with a number of the observations in this study. First, it provides an explanation for the basic phenomenon in which different elements of the lung are recruited at 20 cm H2O. It may also explain the subsequent changes in lung mechanics observed following inflation beyond 20 cm H2O. If these regions become unfolded during an inflation, then the fluid in these folds is likely to be distributed more evenly throughout the alveolus. Under these circumstances these regions are likely to unfold at lower Prs in subsequent maneuvers, resulting in a more compliant lung.
Although it is possible that a number of mechanisms could be responsible for these observations, it is clear that fundamental changes in the mechanical properties of the mouse lung occur at a transrespiratory pressure around 20 cm H2O. Further structural and functional studies are warranted to clarify the mechanisms involved.
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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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