INTRODUCTION

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IT IS GENERALLY AGREED THAT over a wide range of lung volumes (VL), the architecture of the mammalian lung displays approximate geometric similarity. However, the design of the lung includes an internal degree of geometric freedom in that the division of acinar volume between alveoli and alveolar ducts is determined by a balance of forces: inward-acting tissue forces in the alveolar entrance ring and outward-acting forces arising from septal tissue forces in parallel with surface forces at the air-liquid interface (3, 12, 27). This implies that alveolar surface area (S) can change independently of VL as a consequence of changes in the surface tension () acting at the air-liquid interface of the alveolar septal surface. There are wide variations in , both in normal lungs (especially true for large-volume maneuvers but also true for tidal breathing) and in diseased lungs (such as in acute respiratory distress syndrome), and therefore it is important to assess the magnitude of variations in surface area associated with these changes and their physiological consequences to lung recoil (PL) and gas exchange.

The interplay between surface forces and tissue forces on the one hand and radially inward- and outward-acting forces on the other hand is shown diagrammatically in Fig. 1. The effect of increased  is seen to have two effects. First, overall PL is increased, and second, S of the alveolar septa, especially those with free edges, is decreased. It follows that the tissue forces within those septa (excluding the free edge) are correspondingly reduced and the tissue forces in the free borders of the septa are increased; the net effect is that the increase in PL is less than that which would have occurred had there been no change in S. Furthermore,  itself is not an independent variable insofar as it is strongly dependent on both the surface area of the surfactant film as well as the inflation-deflation history. Thus the simplest measure of lung mechanics, i.e., the pressure-volume (P-V) curve, depends on the simultaneous solution to the geometric response to changes in , the response of  to changes in S, and the interaction with tissue forces both within the septa and at their free edges.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Cartoon [modified from Wilson and Bachofen (27)] of the interplay of forces between the alveolar septa (comprising both tissue and surface forces) and the alveolar duct (elastic tissue and smooth muscle in the alveolar entrance rings, depicted as springs). As shown, lowered surface tension () results in ductal retraction and increased surface-to-volume ratio (S/V); conversely, increased  results in septal retraction and lowered S/V.

In this study, we determined the fractional change in S (S/S), at the same VL, between control conditions and after changing . We then determined the relationship between S/S and the change in PL (P) caused by changes in . This was done over a range of VL from functional residual capacity (FRC) to total lung capacity (TLC, the volume at 30 cmH₂O distending pressure). All measurements were made in fresh excised rabbit lungs, by use of diffuse light scattering technology that, because the lungs are not fixed, permits repeated examination of an individual lung both at different volumes, and before and after altering .  can be altered by lavage with a variety of fluids, followed by reinflation with air [e.g., Smith and Stamenovic (20), Bachofen et al. (1), and Hoppin et al. (13)], but this is known to leave a significant amount of fluid in the lung (4). The septa of partially filled alveoli are thus not equivalent to two-dimensional membranes with (altered)  at the air-liquid interface. Thus, in the lavage preparation, the alveolar architecture is changed owing both to the direct effects of changes in  and to partial alveolar flooding. We therefore chose to alter  by ventilating the lung with nebulized polydimethylsiloxane (hereafter abbreviated as siloxane), the volumetric dose of which was several orders of magnitude less than techniques employing lavage. We found that the shifts in the pressure-volume characteristics with exposure to nebulized siloxane were remarkably similar to those found in air-filled lungs following lavage with the same fluid (20), a technique generally thought to effect an approximately constant  preparation (13, 28).

Using these techniques, we found that S/P systematically decreased with increasing VL. Near FRC it was about half that concluded by Wilson (26), using data of Bachofen et al. (1) and Gil et al. (9) obtained from fixed lungs. At high VL, S/P was very close to zero.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Two groups of New Zealand White rabbits were used for these experiments: one group (n = 9) for characterizing the effects of nebulized siloxane on pulmonary mechanics (pressure/volume curves, residual siloxane, and regional variability); the other group [n = 7, surface-to-volume ratio (S/V) study] for measuring the effect of alterations in , as quantified by changes in PL, on pulmonary surface area.

Effects of nebulized siloxane. The deflation P-V characteristics were studied after three different types of exposure to constant- siloxane. In exposure type 1 (repeat nebulized siloxane, n = 3), lungs were excised and control P-V data were collected. The lungs were then ventilated with nebulized siloxane, and posttreatment P-V data were collected. Ventilation with nebulized siloxane and P-V data collection were then repeated two more times. In exposure type 2 (nebulized siloxane vs. lavage, n = 3), excised lungs were studied under control conditions, after a single nebulized siloxane treatment, and after siloxane lavage and reinflation with air. In exposure type 3 (nebulized siloxane in vivo, n = 3), anesthetized animals were ventilated with nebulized siloxane, the lungs were excised, and postnebulization P-V data were collected.

Animal preparation. Each of the 16 animals (mean body wt 3.7 kg) was anesthetized with ketamine (30-50 mg/kg im) and xylazine (1-3 mg/kg im). Additional pentobarbital sodium (10-20 mg/dose iv) was given via an ear vein as needed to maintain adequate anesthesia. Thirteen rabbits (treatment types 1 and 2 and animals used for the S/V study) received heparin (1,000 units/kg) and were exsanguinated via the carotid arteries. They were then intubated, the trachea was clamped at positive airway opening pressure (Pao), and the lungs were excised. Three rabbits (treatment type 3) were intubated below the larynx through a midline incision and ventilated with nebulized siloxane. The animal then received heparin (1,000 units/kg iv) and was exsanguinated via its carotid arteries. The lungs were then excised.

Experimental setup and ventilatory histories. Each pair of rabbit lungs was suspended by the trachea, and the endotracheal tube was connected to an adjustable constant-pressure source for control of airway opening pressure (Pao). The lungs were kept moist with 0.9% saline. Between measurements, the lungs were ventilated at 20 breaths/min (Harvard respiration pump, Harvard Apparatus, Millis, MA), and during measurements they were held at a constant airway pressure. The ventilator was operated as a "pressure ventilator" with the addition of pop-off valves to its inspiratory and expiratory limbs. The pop-off valve on the ventilator's inspiratory limb was set to open at Pao  25 cmH₂O, whereas its passive expiratory limb's valve was set to open at positive end-expiratory pressure of 2-3 cmH₂O (pretreatment) or 7-10 cmH₂O (posttreatment). This increase in positive end-expiratory pressure posttreatment was done to prevent atelectasis and maintain FRC near control levels, because the siloxane treatment raises the lung's  and recoil pressure, especially at low VL. Before each measurement, the lung was cycled three times between Pao of 3 and 30 cmH₂O and then set to the target Pao on the lung's deflation limb. During measurements, Pao was held constant by connecting the lung to an air source with a "T" side branch submerged to a measured depth in a water column. Because there is no gas flow during measurements, Pao is a direct measure of PL. At each target Pao ranging from 2.5 (pretreatment) and 5 (posttreatment) to 30 cmH₂O, both VL and S were measured, as described below.

VL measurement. At PL ~10 cmH₂O, three pleural markers (short pieces of black suture) were cemented to the middle portion of the costal pleural surface of one diaphragmatic lobe (S/V experiments) or both left and right lobes (siloxane-treatment experiments) by use of a small drop of cyanoacrylate glue, ~1.5 cm apart in a triangular pattern. The edge lengths were measured either with calipers (for the S/V experiments) or from photographs of the pleural markers together with a ruler held parallel to the lung surface (siloxane-treatment experiments). The area of the triangle was computed from the edge lengths. VL was calculated as the 3/2 power of the area inscribed by the suture markers (14), normalized to its value at TLC pretreatment. (TLC is defined as VL at Pao = 30 cmH₂O). Note that VL as thus defined is an estimate of the sum of gas and tissue volume. Pressure and volume data were fitted by least squares to the exponential expression of Salazar and Knowles (18)
where V₀ is VL at zero transpulmonary pressure, VC is the change in volume from V₀ to its asymptotic value at high transpulmonary pressures (essentially equivalent to 30 cmH₂O), and P₀ is the pressure at which VL reaches 69% of its asymptotic value from the PL = 0 state.

S measurement. Fractional changes in S, or, equivalently, surface-to-volume ratio, were determined by using diffuse scattering of laser light (693 nm). These changes were measured, during control conditions, with TLC used as the reference state. After nebulized siloxane treatment, measurements were repeated, using pretreatment isovolume points as the reference states. For a description of the theories, techniques, and interpretations involved when using diffuse light scattering in measuring lung architecture, see Butler et al. (6, 7), Suzuki et al. (21), Miki et al. (16), and Topulos et al. (22). Briefly, one optical fiber was used to place a point source of light normal to the pleural surface of the lung lobe, and three receiving fibers detected the backscattered light intensity at fixed and known distances from the optical source. In the diffuse far field of scattering, the intensity falls off with distance r from the source proportionate to (k_diffL_opt/r²)exp(k_diffr), where k_diff is the diffuse extinction coefficient and L_opt is the optical mean free path for scattering. Fitting a linear function to the logarithm of the r²-weighted intensity suffices to determine k_diff, the logarithm of k_diffL_opt, and hence the fractional changes in L_opt from any reference state. These can be converted to fractional changes in surface area by observing that L_opt is proportional to L_m² (21), where L_m is the mean linear intercept of air-liquid interfaces in the lung and that L_m is proportional to (S/V)¹, i.e., the inverse of S/V.

Treatment of the lungs with nebulized siloxane. After control measurements, the lung was ventilated for 5 min at 20 breath/min. Treatment was then begun with nebulized siloxane (5 ml polydimethylsiloxane; viscosity 20 cS, molecular weight = 2,000,  20.6 dyn/cm; Nye Lubricants, New Bedford, MA) in the inspiratory limb of the ventilator by using a continuous flow of ~13 l/min compressed air into the nebulizer (Misty-Neb, Allegiance, McGraw Park, IL). The time necessary for nebulizing the entire 5 ml of siloxane was between 5 and 10 min. Gas leaving the circuit during nebulization passed through HEPA filters (OF612HE Omega filter, Atrix International, Burnsville, MN) to avoid contaminating the room with siloxane aerosol. The size distribution of the siloxane aerosol leaving the nebulizer was characterized (Aerosizer, model MACCII, API, Amherst, MA). The count median aerodynamic diameter of the mist leaving the nebulizer was 1.8 ± 1.3 µm (mean ± geometric SD). After treatment, the lung was ventilated for either 5 min (siloxane-characterization measurements) or 30 min (S/V measurements).

Siloxane lavage. After control data and data after a single nebulized siloxane treatment were obtained (as above), the lungs of rabbits in the type 2 exposure group (nebulized siloxane vs. lavage) were lavaged with the same fluid. The lungs were filled by connecting the trachea (starting from a PL of 0) to a siloxane-filled reservoir held 30 cm above the lung. Filling required 80-88 ml of siloxane and took ~15 min. To drain the siloxane from the lungs of two animals, the reservoir was then lowered to ~100 cm below the level of the lungs. The siloxane was withdrawn from the lungs of the third animal by aspiration with a syringe and passive drainage. After 15-20 min, 22-25 ml of siloxane remained in the lungs. The lungs were then ventilated with air for ~5 min, and then postlavage P-V data were collected as described above.

Siloxane dose deposited. In separate experiments, the volume of siloxane deposited per gram of lung tissue was determined in three excised rabbit lungs and one excised dog lobe ventilated with nebulized siloxane and in one rabbit lobe ventilated in vivo with nebulized siloxane. The mass of elemental Si in each of 23 samples (1.3 ± 0.5 g) was determined by atomic absorption spectroscopy (Galbraith Laboratories, Knoxville, TN). Also analyzed were untreated control lungs prepared in an identical manner, to which known volumes of siloxane were added after homogenization (used to calculate Si recovery efficiency). The volume of siloxane deposited per gram of lung tissue was computed from recovery efficiency (~0.63), the density and composition of the siloxane, and the mass of each sample.

	RESULTS

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Effects of siloxane exposure on the P-V curves. The P-V characteristic of the lung changed consistently after all of the exposures to siloxane, with no important differences between a single nebulization, multiple nebulizations, or lavage. Figure 2 shows a comparison of P-V curves under control conditions; one, two, or three treatments with nebulized siloxane; and after lavage. Elastic recoil increased between ~40-90% TLC. At higher VL there was less change in the P-V curves. Regardless of the exposure method, the magnitude of the change in recoil at 50% TLC was always ~5 cmH₂O. These P-V findings are similar to those of Smith and Stamenovic (20) in rabbit lungs inflated with air after lavage with the same siloxane, as shown in Fig. 3.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Mean of all deflation pressure-volume (P-V) data in lungs used for siloxane exposure characterization, pooled by siloxane exposure. For clarity, error bars are not shown in this composite figure, but SD [as a % of total lung capacity (TLC)] over the range of lung volumes (VL) studied are given below for each group. , Presiloxane control (n = 6; SD = 4%). , , and : After 1, 2, or 3 exposures to nebulized siloxane (respectively, n = 6, SD = 11%; n = 3, SD = 7%; n = 3, SD = 8%). ×, After a single nebulized siloxane exposure in vivo (n = 3; SD = 7.3%). +, Air-filled after siloxane lavage (n = 3, SD = 11%). PL, lung recoil. In this and subsequent figures, VL is normalized to its pretreatment value at airway opening pressure (Pao) = 30 cmH2O.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Comparison of P-V data (means ± SD) in lungs used for siloxane exposure characterization with the data of Smith and Stamenovic (20). , control (n = 6); , After a single treatment with nebulized siloxane (n = 6). These are the same data as in Fig. 2, with SD bars added. The deflation P-V data of Smith and Stamenovic (20) are shown as , presiloxane control; , air filled after siloxane lavage.

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View larger version (15K): [in this window] [in a new window]   Fig. 4.   Pooled P-V data (means ± SD) in lungs used for lung S/V measurements (n = 7). Exponential fits following Salazar and Knowles (18) are also shown. , Control; , postsiloxane exposure. Note that the two P-V curves cross at a VL somewhat above 90% TLC. Below this point, recoil is increased with treatment, consistent with increased , and vice versa above the crossover volume.

Alveolar S, VL, and P. Pretreatment S varied strongly as a function of VL. Figure 5 shows a log-log plot of S vs. VL (mean ± SE). Control data points are shown, together with the regression line (constrained to go through the origin because both S and VL are normalized to their values at TLC). The slope is 0.65, which is not significantly different from .

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Double logarithmic plot of S (mean ± SE), normalized to its value at TLC pretreatment, as a function of VL. , Control measurements; , after siloxane exposure. Line is a linear regression on the control measurements, constrained to go through the origin (the pretreatment TLC point); log S/S(TLC) = 0.65(±0.03) log VL/TLC.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Changes in S and PL secondary to treatment with nebulized siloxane. The 4 pairs of points (mean ± SE) and solid lines represent data at the indicated VL. Corresponding to these data, we also show, with the dotted lines, data derived from Wilson (26) from primary data of Bachofen et al. (1) and Gil et al. (9). These lines are drawn from our measured values of surface area in control conditions (P = 0), with slopes given by the chord slopes (air to detergent) for VL from 40 to 80% TLC, and by the tangent slope at TLC, in Fig. 1 of Wilson (26). The lines are terminated at our measured values of P posttreatment. Note that at all VL the change in S was significantly smaller in the present study than the results of Wilson (26).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Fractional change in S (pre to post) as a function of change in lung recoil (P). Here each point corresponds to data taken at the indicated VL. The present results are displayed as means ± SE, solid line. Also shown are data from Wilson (26) (dotted line) interpolated to the same values of P posttreatment that we observed.

	DISCUSSION

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Our measurements of the change in parenchymal surface area, at fixed volumes when  is artificially changed, are similar to but quantitatively smaller than that seen by others [Bachofen et al. (1) and Gil et al. (9) as synthesized by Wilson (26)]. There are two differences in the experimental preparations that may account for this. First, our measurements were performed in fresh, unfixed lungs by use of diffuse light scattering, whereas the classical measurements of surface area have been done in fixed and sectioned lungs by using stereological principles and morphometric measurements made under light microscopy. Second, our intervention to change the lung's  involved ventilation with nebulized siloxane, which resulted in a deposited volume significantly smaller than is seen with previous lavage techniques. We remark on each of these. After these methodological issues, we give some interpretations of our findings in terms of their physiological and pathophysiological implications.

How are forces "fixed" when chemically preserving a lung? The inwardly acting forces in the tissue comprising the alveolar entrance rings are essentially at all times balanced by the combination of outwardly acting tissue forces in the alveolar septa together with the  present at the air-liquid interface. For the resulting geometry (fractionation of volume between ducts and alveoli, or, equivalently, the determination of the alveolar septal area at that given VL) to remain unchanged during fixation requires that the forces that are mechanically in series change quantitatively in an identical fashion over time. If this is not the case, then there will be a distortion of the geometry during fixation before the final state is reached when sections can be cut. The change in the force/length characteristics of connective tissue (primarily elastin and collagen) as well as smooth muscle during aldehyde fixation is not well known. Similarly, there are no studies to our knowledge of the time course of changes in  in the lung (be they associated with native surfactant or with any of the perturbing fluids or detergents used to artificially change ) during vascular perfusion with osmium tetroxide or glutaraldehyde. Moreover, leakage of any fluid into the alveolar space may transiently increase  before fixation and result in a larger decrease in surface area. The question of whether there is geometric distortion during fixation, particularly as manifested in changes of surface area relative to that present in vivo, thus remains open. It is important to note that we are not questioning the value of classical fixation techniques in preserving the ultrastructure at the cellular and subcellular level, nor do we question the appropriateness of using stereological techniques to infer three-dimensional properties such as S/V from morphometric measurements made on sections. Rather, it would seem important in the future to characterize the temporal evolution of stiffening of proteins and surface films in the presence of aldehyde perfusates. This would resolve the possibility that differences in our data, obtained on fresh, unfixed lungs, from classical studies are due to architectural alterations in the lung parenchyma associated with perfusion fixation of prestressed tissues.

Diffuse light scattering. In addition to the uncertainties associated with chemical fixation, there are also a number of unanswered questions regarding the use of diffuse light scattering to obtain stereological information. Chief among these is the relationship between fractional changes in the optical mean free path (which we feel confident we can measure) and the corresponding fractional changes in the geometric mean free path (which is the desired quantity, inversely proportional to S/V). The relationship between the optical and geometric mean free path was established by Suzuki et al. (21), by comparison with microscopic morphometry on preparations fixed by vascular glutaraldehyde and osmium tetroxide perfusion, assuming that the fixed preparation is reasonably faithful to the in vivo architecture (17). It is important to note that the measurements we report here are expressed as fractional changes in S/V, not absolute values. We therefore cannot make any direct comparison with that portion of Wilson's work that quantifies the specific relationship between  and S/V. Furthermore, we have explicitly extrapolated the calibration of Suzuki et al. (21) to the posttreatment preparation; the magnitude of the error involved here is unknown, but we suspect that it is small, because films significantly less than a wavelength of light in thickness have little effect on the optical properties of surfaces. On the other hand, an upper bound on this effect can be calculated in a manner similar to that in Butler et al. (6), treating the coated septa as an optical composite (siloxane-tissue-siloxane; index of refraction 1.4:1.33:1.4). The scattering cross sections with and without the siloxane film present amount to ~5%, which we take as negligible. In summary, even in light of the above uncertainties, diffuse light scattering has the singular advantage of being applicable to fresh, unfixed lungs, and, in consequence, individual preparations can be used as their own controls, either for comparison at different VL (see Fig. 5) or for comparison at a variety of isovolume points before and after treatment (see Figs. 6 and 7).

Altering the lung's . There is little doubt that treatment with nebulized siloxane significantly alters the  in the lung. This could result from the siloxane spreading into a thin and continuous film at high VL, where the surfactant  is greater than 21 dyn/cm, and, through unknown mechanisms, remaining spread at low VL, even though that is not the energetically favored state. In this case, the lung would have an approximately constant , independent of VL. By contrast, it is also possible that the siloxane contaminates the entire surfactant film sufficient to raise  well above normal values at low VL, although not necessarily to the siloxane-air interfacial value. This possibility is supported by the observation that contamination from protein leakage in diseases characterized by increased capillary permeability can lead to significant surfactant dysfunction, with elevated . In this case there is no reason to believe that  posttreatment is constant. We have no experimental evidence to resolve this question, which must therefore remain open. Nevertheless, it is important to recognize that, whether or not  is constant, exposure to nebulized siloxane results in consistent changes in PL, and the values we report here for the change in surface area per change in recoil pressure would still be valid, insofar as this ratio is an estimate just of the tangent S/PL|_VL. In particular, the specific value of  does not enter into our calculations; in this sense one can consider the "dose" in our treatment to be the change in PL, albeit through the agency of the nebulized siloxane.

Physiological significance. In his investigations into the relationships among PL, surface area, and , Wilson (25, 26) concluded that at any given VL, increased  leads to an increase in PL through both its direct effects [quantitatively given by P = ()S/V] and its indirect effects via a net increase in ductal tissue forces due to their serial arrangement with alveolar septa. (This is true independent of parenchymal geometric distortion, but note that decreases in S will reduce tissue forces within the septa, because they are in parallel with the septal surface.) An equivalent expression of this phenomenon is to observe that, in the absence of geometric distortion, an increase in  would lead to increased PL in excess of that which is observed; the presence of distortion allows the lung to relax to a new equilibrium state at a lower PL. The extent to which this occurs can be quantified by the change in surface area with changes in PL under isovolume conditions. The fact that we found less area change with changes in recoil then implies that alterations in  such as found in disease would be expected to have a more serious impact on PL than would have been predicted. Indeed, the smaller the change in area with PL, the closer P is to the observed P.