Veterans Affairs and Duke Medical Center, Durham, North Carolina 27705
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ABSTRACT |
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Mechanical ventilation of the lung could affect surfactant turnover by alteration of its secretion, recycling, and degradation. In vitro studies of surfactant subfractions recoverable from lavage fluid have led to predictions about surfactant physiology in vivo that include morphological transformations. We used electron microscopy to study in situ lipid forms in alveoli of rat lungs after two ventilation strategies [15 min at pressures (cmH2O) of 20/0 or 20/10]. In control animals, 4% of the lipid profile area in the surface lining layer was myelin figures (MF), 14% was tubular myelin, 37% was vesicular forms (VF), and the remainder (45%) was hypophase. Compared with controls, the length-normalized sum of the lipid forms and the hypophase was two times as great in the lungs of the 20/0 group. MF were threefold higher in the 20/0 group and fivefold higher in the 20/10 group. VF doubled after ventilation at 20/0, but VF were the same as control after ventilation at 20/10. The results showed that a ventilation pattern of 20/0 compared with that of 20/10 group was associated with a significantly larger VF, suggesting an increased net production of these surfactant forms during a large tidal volume breathing pattern. These morphological results are consistent with published results using physical methods of fractionating lung lavage.
myelin figures; tubular myelin; vesicular forms; electron microscopy
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INTRODUCTION |
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MECHANICAL VENTILATION of normal lung is a frequently required and widely used method of patient support in clinical practice. The question whether certain mechanical ventilation regimens could result in tissue injury of previously normal lung has significant clinical importance. It has been described that large tidal volume (26), high inspiratory and expiratory pressures (23), long duration of ventilation (20), and oxygen at high inspired partial pressures (4) can cause lung injury. The several mechanisms causing lung damage have not been completely understood; we cannot recognize very early changes of lung injury, and our ability to prevent these injuries is limited. Surfactant inactivation has been suggested to play a role in causing lung injury, especially after a period of large tidal volume breathing (24, 35). Early recognition of surfactant inactivation might improve clinical detection of incipient lung injury (13).
Surfactant obtained by bronchoalveolar lavage has been separated by centrifugation into several subfractions as described by different authors. The P3 [1,000 average gravitational force ( gavg)] and P4 (10,000 gavg) subfractions of lung lavage isolated by Magoon et al. (14) and the ultraheavy and heavy fractions of lung lavage isolated by density gradient centrifugation by Gross and Narine (5, 6) were rich in myelin figures (MF) and contained tubular myelin (TM) as judged by electron microscopy. Lewis and co-workers (12) modified an earlier protocol (8) to isolate a lung lavage fraction at 40,000 gavg, named it the large surfactant aggregate, and reasoned that it would be enriched in MF and TM.
The P5 subfraction of Magoon et al. (14), isolated at 100,000 gavg, and the light subtype described by Gross and Narine (5, 6) were demonstrated by electron microscopy to be rich in vesicular forms (VF). A fraction not pelleted by 40,000 gavg was named small surfactant aggregates by Lewis et al. (12, 13).
A metabolic relationship between these surfactant subfractions was proposed (14) in which the most dense and most aggregated lipid forms, MF and TM, are considered the product of secretion and unfolding of type II cell lamellar bodies. The VF may be a "used" or "spent" form of surfactant arising after desorption of lipid from the interfacial film (14). Alternative relationships have been proposed by Thet et al. (22). Fractions containing the large and dense surfactant forms, presumably rich in MF and TM, can reduce surface tension during compression (19) and will increase lung compliance when administered to the lungs of surfactant-deficient animals (36). The least-dense subfraction adsorbs very slowly to an air-liquid interface and has little surface activity in vitro or in vivo (14).
Cycling of the alveolar interfacial film during large tidal volume excursions might be expected to 1) result in a measurable release of lamellar bodies from the intracellular pool and 2) increase the rate of exchange of MF and TM through the surface and into a recycling pool. Several in vivo (16, 18, 35) and in vitro (6, 31) studies suggest that stretch will stimulate secretion, that small lipid vesicles are formed during cycling of the interfacial film, and that depletion of a precursor pool can occur and can lead to a lack of adequate surface tension-lowering material.
Alveolar lipid material obtained by lavage could be contaminated by components from airway mucus or by serum factors from vascular compartments, and because artifacts could be generated during isolation, a test of the predictions of the biochemical studies using a direct morphological method was done. By using semiquantitative electron-microscopic methods, we estimated the relative pool sizes of ultrastructurally recognized surfactant forms and examined the effects of two ventilation strategies on the ratio between surfactant forms.
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MATERIALS AND METHODS |
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Animals and ventilation regimens. Fifteen specific pathogen-free male Sprague-Dawley rats (Charles River, Raleigh, NC) weighing 290-535 g were used in this study. The animals were anesthetized with intraperitoneal 100 mg/kg pentobarbital sodium. A tracheotomy was performed, and the trachea was cannulated and connected to a pressure-limited rodent ventilator (Harvard, South Natick, MA).
One group was control animals whose lungs were fixed inflated immediately after tracheostomy. A second group (20/0) included animals ventilated for 15 min at 50 breaths/min, inspiratory pressure of 20 cmH2O, and no positive end-expiratory pressure (PEEP). The third group (20/10) was animals ventilated for 15 min with frequency of 50 breaths/min, inspiratory pressure of 20 cmH2O, and 10 cmH2O PEEP, which was applied by submerging the expiratory line under water. To record the tidal volume resulting from these ventilatory regimens, we collected the exhaled air from 50 breaths of one 300-g animal ventilated first at 20/10 and then at 20/0. The average of five trials was 2.9 ml (BTPS) at 20/10 and 9.0 ml at 20/0.
At the end of the ventilation period, the animals were exsanguinated, and the thorax was widely opened with the lungs kept statically inflated with air at ~20 cmH2O while the pulmonary artery was cannulated. Fixation was done by perfusion of the inflated lungs with 2% glutaraldehyde in 0.085 M sodium cacodylate buffer, pH 7.4, followed by 2% OsO4 in the same buffer.
Tissue processing. Specimens of lung tissue were taken from the apical, middle, and lower portions of the left lung, and 1- to 2-mm tissue cubes were cut. These tissue cubes were mixed and washed in cacodylate buffer, pH 7.4, postfixed with 2% OsO4 overnight, stained with 2% uranyl acetate for 90 min, and dehydrated in a graded series of cold acetone. Next, 10 cubes were arbitrarily chosen and were embedded in Poly/Bed 812 (Polysciences, Warrington, PA; see Ref. 27). Two blocks of embedded tissue were arbitrarily selected from each lung, and four ultrathin sections (of ~65 nm thickness) from every block were cut with a diamond knife on a Reichert-Jung ultratome. The sections were placed separately on Formvar-coated 200-mesh Cu/Rh grids. Double staining was performed with 4% uranyl acetate for 20 min and with lead citrate for 2 min. Grids were viewed and photographed with the use of a JEOL 1200 electron microscope.
Morphometry. Four squares of every grid were examined completely. The examination included a low-magnification micrograph (taken at ×600 magnification and printed at ×2,100 on 11 × 14-in. paper) of one whole grid square and micrographs (taken at ×15,000 and printed at ×40,000 on 8 × 10-in. paper) of any visible surfactant material.
An illustration of different extracellular surfactant forms is shown in
Fig. 1. Myelin-like forms, MF, were
identified as those profiles that consisted of large (0.5-1.5
µm) forms with multiple concentric lipid layers. They often were
found near the apical surface of the type II cells. MF were often
associated with TM, and sometimes MF reached the air-liquid interface.
The TM profiles had a characteristic latticelike appearance and were easy to recognize. In most cases, TM profiles were localized just underneath the continuous layer representing the air-liquid interface. We labeled unilamellar vesicles as VF. Most of the VF were small and
uniform in size, ranging from ~0.05 to 0.10 µm. They usually were
found in the hypophase layer but could be found anywhere within the
lining fluid. We also identified as VF those larger (0.5 µm)
unilamellar vesicles that were seen occasionally. Our morphological
criteria for identifying hypophase were its relatively homogeneous
staining and its lack of formed elements.
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The boundaries of the profiles were manually traced from the ×40,000-magnification micrographs with a cursor on a digitizer table (GTCO, Columbia, MD). The traced boundaries for each profile were analyzed with the use of a commercial software package (Sigma Scan; Jandel Scientific, Corte Madera, CA), and the enclosed area was calculated. No attempt was made to trace an area representing the extensive interfacial film, which appeared as a monolayer at the magnification we used, because this image was too thin to trace. To control for differences in the total area of alveolar surface studied in each animal group with length-weighted data, we also traced on ×2,100-magnification micrographs the line of the alveolar epithelial surface (e.g., the luminal plasma membrane of the type I and type II cells), which represented the entire surface examined for each section.
Calculations. From the digitization,
we obtained two types of data: the directly measured area of surfactant
form profiles, psf
(mm2), and the length of the
alveolar epithelial surface, l (mm). The sums of the areas,
(PSF;
µm2) and lengths
(L; mm) were calculated as follows:
PSF = psf/mag2
and L =
l/mag, where mag is magnification
(Table 1). The length-weighted data (WSF;
µm2/mm) for the amount of each
surfactant form found on every examined grid square as well as the mean
for the grid and the totals for each animal were calculated using the
simple formula
WSF = PSF/L. The mean results for each group were determined by dividing
WSF by the number
of animals (Table 2).
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Statistical analysis. The nonparametric rank sum test was used to compare the results obtained from the three groups of experimental animals (controls, 20/0, and 20/10). Comparisons were done between area-weighted values for the animals and also between percent values for the areas of different forms (MF, TM, and VF).
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RESULTS |
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Control animals. We traced 464 µm2 of profile boundaries that
were distributed beneath an interfacial film that lined alveolar walls
with a total estimated length of 11.6 mm (Table 1). MF occupied 4%, TM
was 14%, VF represented 37%, and the rest was hypophase, 45% of the
area traced. Data normalized to the alveolar length for every animal in
the control group are shown in Table 2. The control group total for the
length-normalized lipid-rich material (the sum of MF, TM, and VF) was
21.9 µm2 · mm1 · animal
1,
whereas the hypophase was 17.1 µm2 · mm
1 · animal
1.
Ventilation group 20/0. For the
animals exposed to this large tidal volume regimen of ventilation, the
total amount of profile boundaries traced was 741 µm2 distributed over 10.9 mm of
alveolar length (Table 1). The percentage distribution of total
surfactant profile boundaries was as follows: MF 5%, TM 11%, VF 37%,
and hypophase 47%. Results for individual animals and the average for
the group after the length normalization are summarized in Table 2. The
three lipid-rich forms (MF, TM, and VF) had an average normalized value
of 44.2 µm2 · mm1 · animal
1.
The measured hypophase was 40.6 µm2 · mm
1 · animal
1.
Unique to this group, we found regions within the alveolar surface that
were rich with VF. We also found areas where a row of vesicles on the
alveolar air side of the interface was observed instead of the usual
unique lipid layer (Fig. 2).
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Ventilation group 20/10. Animals from
this group had 744 µm2 of
surfactant profile boundaries over the 11.5 mm of alveolar wall we
studied (Table 1). The distribution between the different morphological
compartments was as follows: MF 11%, TM 12%, VF 23%, and hypophase
54%. Individual and group data are summarized in Table 2. The average
value for the lipid-rich forms was 31.6 µm2 · mm1 · animal
1.
The hypophase was 36.0 µm2 · mm
1 · animal
1.
There were areas where multilayers of lipids arranged parallel to the
surface film could be seen. These ordered layers were separated by an
approximately equal distance similar to TM. On occasion, these ordered
layers appeared connected with TM profiles (Fig.
3).
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Comparison between controls and 20/0.
The amount of normalized total surfactant profiles was two times as
large (118%) as control in those animals that were exposed to the 20/0
ventilation strategy (Table 2). Figure 4
shows that the average value for MF was nearly 3-fold greater than
control, TM was 1.3-fold larger, and VF was more than doubled. The
hypophase was also two times larger in the 20/0 ventilated animals
compared with controls.
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Comparison between controls and 20/10. We found 73.5% more normalized total surfactant lipid profile area in the PEEP group than in controls. Compared with control animals, the 20/10 group had fivefold greater normalized values for MF and also slightly higher (1.45-fold) amounts of TM profiles. VF were nearly the same after this ventilatory regimen compared with controls, but the hypophase volume was two times as large (Fig. 4).
Comparison between 20/10 and 20/0. The 20/0 group had about one-half the amount of MF with the same quantity of TM and more than two times the VF compared with the positive pressure (20/10) group (Fig. 4). In both groups, the hypophase was equally higher compared with controls, nearly twice.
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DISCUSSION |
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The alveolar lining layer has a bimodal distribution, as it is very thin over most of the alveolar wall but is relatively thick within the alveolar corners and clefts (3). After vascular perfusion fixation of the lung and processing designed to enhance lipid retention, we found apparently well-preserved surfactant lipid forms. The morphological results of the preservation scheme we used are qualitatively very similar to the results of freeze fracture without fixation (3, 15, 28). An independent test could not be done of whether any quantitative alterations in the lipid forms of the alveolar liquid were caused by our fixation techniques.
We randomly sampled the alveolar surface and identified the surfactant lipid forms shown in Fig. 1. We measured the relative volumes of three distinct surfactant morphologies in those areas where accurate tracings could be done at the final magnifications we chose. These measurements were mainly at those areas where the surface layer was relatively thick. Over the long midseptal portions of the alveoli, there were areas with scanty surfactant material, and there were many zones where we found only an osmiophilic layer at the air-liquid interface. This single layer was either bound to the alveolar surface of type I cell plasma membrane or lay slightly away from the cells. We did not measure the volume of surfactant in the midalveolar zones, where the images were just a single line, but we did trace the alveolar length of those regions.
From our data for the control group, an average thickness of 0.04 µm of the surfactant subphase covering the air surface side of alveolar epithelial cells could be calculated. Bachofen et al. (1) reported more than two times higher volumes of the hypophase when normalized to the unit capillary endothelium basement membrane, and Bastacky et al. (3), using low-temperature scanning electron microscopy, found an average of 0.20 µm. A lower estimate from our data was expected because we did not include the volume of the thinnest extensive regions of the extracellular lining material, and our fixation techniques would not be expected to preserve a protein-poor water phase as well as could be accomplished with the freezing method used by Bastacky et al.
We found a twofold larger extracellular layer after both ventilation strategies, consistent with either a secretory response to mechanical ventilation, an accumulation of fluid due to inflation of the lung, or some combination. The normalized average volume of lipid-rich figures (MF, TM, and VF) doubled after the 20/0 ventilatory regimen, but with added PEEP (20/10), the increase was much smaller (44%).
MF may represent a product of exocytosis by type II cells. Their intracellular precursors may be lamellar bodies that contain the surfactant lipids. We considered MF as a pool that could be enlarged due to increased secretion from type II cells or due to slowed transformation into TM or into the air-liquid interface monolayer. We cannot choose between these possible mechanisms. The enlargement of the MF pool in both groups of ventilated animals might be the result of an increase in secretion, since the peak inspiratory pressures used should result in significant alveolar distension, which in turn should be a secretory stimulus (18). The MF pool was larger after the 20/10 pattern of ventilation compared with the 20/0 pattern. The difference between the 20/10 group MF and control MF was statistically significant, not only comparing normalized values but also as a percentage of total lipid-rich figures. Because PEEP reduced the areal cycling of the interfacial film, we believe there could have been a greater net MF pool of the 20/10 group due to decreased transformation of MF into the interfacial film.
TM is a highly organized lipid and surfactant protein (SP) matrix formed in the shape of a three-dimensional lattice (25, 29). TM may be derived from MF, and TM may undergo conversion to feed the surface-active film (10, 33). SP-A and SP-B have been shown to have an important role in the formation of TM (30), and it has been recently reported that mice genetically altered to produce no SP-A (SP-A "knockout") form a negligible amount of TM (11). Therefore, the size of the TM pool may reflect not only the amount of alveolar surfactant lipids but also the amount of lipid-bound SP-A in the surfactant layer. What we found was that the TM pool stayed relatively stable and changed a relatively small amount (30-45% and up) after the two ventilation strategies used.
It appears that the amount of TM is not determined only by the quantity of MF in the lining layer. The high stretch-low surface cycling group (20/10) had nearly two times the amount of MF compared with the high stretch-high surface cycling group (20/0) but had the same pool size of TM. Because the amount of any pool is a net balance of influx and efflux rates, the relatively small changes in TM suggest that an approximate balance did exist, although we have no measure of the actual flux rates through the TM compartment.
VF are thought to consist mainly or only of lipids, and they may be formed during the surface film's desorption to the hypophase (32, 34). A serine protease (surfactant convertase) is reported to be required for the transformation of the surface film into surface-inactive small VF (7). The VF may be one form of surfactant ready for recycling back into type II cells (34). An increase in VF could be interpreted as increased turnover (especially degradation) of the surface film or lowered recycling by type II cells.
After the 20/0 ventilatory regimen, the VF pool was larger, but the 20/10 regimen resulted in a VF fraction with the same volume as VF in control lungs. Our data for VF as a percentage of total lipid-rich figures in the 20/0 group are statistically significantly different compared with the 20/10 group. This result is consistent with a key prediction of the hypotheses about extracellular surfactant lipid metabolism.
The hypophase is aqueous material situated beneath the surface layer and has few or no structures. It appears by electron microscopy as an amorphous, homogeneous material that fills the space between the lipid-rich forms and creates continuity of the alveolar lining layer. Hypophase is the place where alveolar macrophages are located. It consists of ions, water, and some unorganized lipids. Other substances like carbohydrates (33), albumin, IgG (9), secretory IgA, and other proteins and peptides (17) that are found in lavage fluid also might be part of the hypophase. Free SP-A may be present in the hypophase, but it has been estimated to constitute <1% of the total SP-A measurable in lavage fluid (2). Although there was an approximately twofold greater normalized amount of hypophase after both of the mechanical ventilation regimens we used, there was no change in the fractional distribution of lipid-rich figures (MF, TM, and VF) and hypophase.
As shown in Table 2, there was substantial variability within our measurements of surfactant components for the individual animals in each group. This may be attributable to the fact that the tissue blocks that we randomly selected represent only a fraction of the total alveolar region. Lung may be heterogeneous, with the result that every small portion of the parenchyma or even each alveolus might have a unique compliance and a unique history of surfactant secretion that would explain significant variability in our analysis of small samples.
What is the mechanism of changes that we observed? One explanation could be the role of different amplitude of the tidal volume during different ventilation strategies. Large tidal volume ventilation produced a high percentage of VF, possibly due to increased conversion of the surface film into VF.
In conclusion, the present in vivo study provides information that two different regimens of pressure-limited ventilation (with and without PEEP) used for a relatively short 15-min period of time affected the morphologically recognized surfactant forms in the alveolar space. Various in vitro schemes for separation of surfactant aggregates from lavage fluid have been offered, and different names for the subtypes have been used (5, 12, 14). Our in vivo study generally validates the in vitro methods.
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ACKNOWLEDGEMENTS |
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We appreciate the assistance of Mariana Tchakalska and Lydia Silbajoris in the digitizing and counting of the micrographs.
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FOOTNOTES |
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This work was supported by the Veterans Affairs Medical Research Service.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Savov, Durham VAMC (151), 508 Fulton St., Durham, NC 27705 (E-mail: jsavov{at}acpub.duke.edu).
Received 30 November 1998; accepted in final form 30 March 1999.
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