Respiration Physiology (1976) 27, 99-l 14;
North-Holland Publishing Company, Amsterdam
EFFECT OF DISTENSION ON RELEASE OF SURFACMNT DOGS’ LUNGS’ ’
IN EXCISED
EDMUND E. FARIDY Department of Physiology, University of Manitoba, Winnipeg, Canada
Abstract. The freshly excised lower lobes of lungs of dogs were washed by filling and emptying the lobe with isotonic saline. The wash fluid was then discarded and the procedure was repeated 9 times. The 9th wash was collected and used for measurements of surface tension and lecithin content. The lobe was then constantly inflated for 3 hr; washed again with isotonic saline (10th wash) and surface tension and lecithin content of wash fluid measured. When the lobe was inflated with air, at room temperature, the surface activity and the lecithin content of the 10th wash were increased in comparison to tbe 9th wash. This increase was not noted when the lobe was inflated with 100 % N, or 100 y0 O2 at room temperature, or with air at 6” C. Increase in the surface activity of the 10th wash was directly related to the inflating pressures used. Thii study suggests that distension of the lung enhances the release of surfactant and that this &ase is a metabolically active process. It appears also that 100% 0, has an inhibitory effect on this pr-s. Lung distension, effect on surfactant Lung lavage Lung metabolism
Oxygen toxicity Pressure-volume Surface tension
measurements, factors afkctmg
In freshly excised lungs, ventilation depletes surfactant, resulting in an increase in lung retractive forces (Faridy et al., 1966). The effects of ventilation are prevented by the application of an end-expiratory pressure and are reversed if the lungs are kept at a constant volume for several hours, provided the temperature of the environment is appropriate and the lungs are not deprived of oxygen at any stage of the experiment. In a metabolically inactive lung (such as dehydrated-rehydrated lung) (Faridy, 1973) the rate of depletion of surfactant by ventilation is greatly increased; positive end-expiratory pressure does not prevent depletion of surfactant ; and the effect of ventilation is not reversed by constant inflation. These observations led us to hypothesize (Faridy et al., 1966; Faridy, 1973)that recovery of the pressur+volume curve in a metabolically active lung occurs as a Accepted for publication 9 March 1976.
’ Preliminary reports of this study have been presented at the meeting of the American Physiological Society: Fed. Proc. (1973) 32: 401. ’ This study was supported by the Medical Research Council of Canada. 99
100
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FARIDY
result of newly formed surfactant replacing that depleted by ventilation, and that distension enhances the replenishment of surfactant. The present experiments were designed to test this hypothesis.
Methods Seventy-three mongrel dogs of either sex, free of respiratory infections, weighing 7.4-12.8 kg, were used for this study. The animals were anesthetized with sodium pentobarbital (30 mg/kg body weight), heparinized and exsanguinated. The lungs were excised; the left and the right lower lobes were separated, and weighed. A plastic cannula was tied into the main bronchus of each lobe, and the pulmonary vessels were ligated. The lobes were then degassed in a vacuum jar, as previously described (Faridy, 1973) and lavaged 9 times. Each lung lavage consisted of filling and emptying the lobe with isotonic saline (5 ml/g initial lobe weight) 4 times from and into a syringe. For the first 2 filling and emptying, the lobe was placed on its mediasnnal surface and for the latter 2 filling and emptying it was placed on its thoracic surface. The wash fluid was then discarded and the procedure repeated 9 times using fresh saline each time. The lobe was weighed prior to the 9th lavage. The fluid from the 9th lavage was collected. The interval between ex~n~n~tion of the dog and the termination of lung ‘lavage #9 was about 60 min. The lobe was then kept at residual volume or at static inflation at predetermined transpulmonary pressures which ranged from 5 to 30 qn H,O. This was accomplished by connecting the lobe to a T tube, one limb of which was kept under water and the other connected to an air tank. The lobe was fully inflated to 30 cm H,O pressure by lowering the limb of the T tube 30 cm under water, then deflated to a predetermined pressure by raising the limb of the T tube to the predetermined depth under water. The lobe was kept constantly inflated by the continuous ,airtlow. The l&es were kept at a highly humidified atmosphere (Mistogen tent) to prevent dehydration. At the end of 3 hr the lobe was weighed and lavaged once again, as described above, with isotonic saline (10th wash). The lobe was not degassed prior to the 10th wash in order to prevent spilling the isotonic saline retained in the lobe after wash 99 As seen in table 1, the volume of air retained in the lung at 0 cm H,O pressure after tish =#9 was small and could have little effect on the distribution of wash fluid in the lung. In addition, most of the trapped air was withdrawn into the syringe during the first and second filling and emptying maneuvers of wash # 10. Fluids from the 9th and 10th washes were collected and used for measurements of surface tension and lecithin content. The results of the 10th wash were expressed as a percentage of the 9th wash to facilitate comparison between experiments. If, during the lung lavage, there was a leak in the lobe or the wash fluid was not clear but light red or red in color (~n~~a~ with blood), such lobes and lung washes were excluded from the study.
DISTENSION
AND SURFACTANT
RELEASE
101
SURFACE TENSION
wash fluid was stirred for 5 minutes by means of a magnetic stirrer, and a 40 ml sample was .&ken. Surface tension was then measured on this sample at room temperature, on a modified Wilhelmy surface tension balance (Kimray-Greenfield surfactometer) (Greentield and Kimmell, 1967) after continuous cyclic compression and expansion of the surface area (between 15 and 100 %) for 2 hr, each cycle lasting 3 min. The lowest surface tension recorded was designated as the minimum surface tension (Y,,,~,,)of each lung washing. In experiments where lung lavage washings were used for surface tension measurements, the lobe after wash 9 wasemptied of isotonic saline until the weight of the lobe was within 1 g of that prior to the 9th washing.
The
AIR DEFLATION PRESSUR-VOLUME
CURVE
The cannulated degassed lobes were attached to the T tube of a pressure-volume apparatus similar to that previously described by Gribetz et al. (1959). The lobe was then inflated with air to 30 cm H,O pressure. This inflation pressure was maintained until the lobe air volume remained constant for 10 sec. The air volume observed at this transpulmonary pressure, considered as total lung air volume (TLV), was designated as 100 % and each volume subsequently observed after deflation to a predetermined transpulmonary pressure (20, 15, 10,5, and 0 cm H,O) was expressed as a percentage of TLV. These pressures were maintained until the volumes were stable for 15 sec. If, during the procedure, the lung air volume did not remain constant at high pressures, air leaks were assumed to be present, and such lungs were excluded from the study. All pressure-volume measurements were performed at room temperature. ISOTONIC SALINE DEFLATION PRES!WRl-VOLUME
CURVE
The cannulated degassed lobe was mounted in an isotonic saline bath, and its bronchus was attached to the T tube of a saline pressure-volume apparatus. One limb of the T tube was connected to a saline-filled burette. The other limb was connected to a vertical tube placed next to the saline bath. The vertical tube, containing saline at the same level as that of the burette, together with a second vertical tube connected to the bath, served as a monometer. By raising and lowering the burette, saline was displaced to and from the attached lo& at pressures simultaneously registered by the manometer. The lobe was inflated with isotonic saline to 12 cm H,O pressure. After stability of the volume at this inflation pressure was achieved, a deflation pressur~volume curve was obtained by lowering the(inflation pressure at decrements of 1 cm II,0 at intervals of 2 min. The saline volume observed at 12 cm H,O transpulmonary pressure, considered as total lung saline volume (TLV), was designated as 100 %, and each volume subsequently observed after deflation to’s predetermined transpulmonary pressure was expressed as a percentage of TLV.
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E. E. FARIDY
If, during the procedure, the lung saline volume did not remain constant at high pressures, or the lobe weight at 0 cm H,O Ptp exceeded the initial lobe weight plus the weight of the saline recorded, saline leaks were assumed to be present, and such lungs were excluded from the study. All press~e-vol~e m~sur~ents were performed at room temperature. LECITHIN
The lung washing was ~ntrifug~ at I650 G for 20 minutes and cell free supematant was then spun at 30,000 x g for 1 hr. The lipids were extracted, from the spun sediment by chloroform-methanol (2 : 1) and washed according to the method of Folch it al. (1957). The sample was dried in a water bath at 40-50 “C under nitrogen and the dried extract reconstituted to 0.5 ml with chloroform. An aliquot of the lipid extract was plated on an activated silica gel-H plate and the lipid fractions separated using a solvent system containing c~orofo~-me~anol-attic acid-water (25 : 15 : 4 : 2) (Parker and Peterson, 1965). The plate was then exposed to iodine vapor and the lecithin spot was identified. After the iodine vapor had evaporated from the plate, the lecithin spot was aspirated into a test tube, the fully saturated lecithin fraction was isolated by mercuric acetate adduction (Mangold, 1961), and the lipid phosphor determined by Brante’s rn~~~tion (Brante, 1949)of the method of Fiske and Subbarow (1925). For calculation of lecithin content in the wash fluid two assumptions were made, namely, that the fluid remaining in the lung prior to the 9th and 10th lavages was in the alveoli and that at the end of lavage it had the same concentration of lecithin as that of the collected fluid. Knowing the inundation of lecithin in the collected fluid, and the amount of fluid retained in the lung (lobe wei~t-~itial lobe weight), the total lecithin content was then calculated. ELECTRON MICR~PIC
STUDIES
Samples of lung tissue were immersed in cold fixative (6.25 % ~u~dehyde in 0.1 M cacodylate buffer, pH 7.4), Sub~~ntly, the lung tissues were transferred into vials containing 6.25 % glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 2 hours at 4 “C. The tissues were rinsed for 24 hours at 4 “C in 0.1 M cacodylate buffer (pH 7.4) containing 0.2 M sucrose (Sabatini et al., 1963). They were then postfixed for 4 hours at 4 “C in 2 % osmium tetroxide buffered in 0.1 M cacodylate (pH 7.4) containing 0.2 M sucrose. Following rapid dehydration, the tissues were embedded in Epon 812 (Luft, 1961).Thick 1 pm sections were stained by R&a&on’s tech& que (Richardson et al., 1960), and examined for general orientation with the light microscope. Thin sections were mounted for electron microscopy on naked 300-mesh copper grids, stained with uranyl aoetate (Luft, 1961) and lead citrate (Reynolds, 1963)and photographed in a RCA EMV 3F electron microscope. A r-test of paired and unpaired variates was empioyed to ascertain differences among the means of two groups of observations.
103
DISTENSION AND SURFACIANT RELEASE
CHARACl’ERIST’ICSOF LAVAGED LUNG
After 9 lung washings the alveolar structure remained unchanged. The distinction between the washed and unwashed lungs was nearly impossible in both light and electron mi~o~pic studies. This was also true in lavaged lobes that had been statically inflated with air for 3 hr and washed for the 10th time, A portion of isotonic saline was trapped in the lung at the end of lavage. The lobe weights, therefore, prior to the 9th and 10th washes were greater by 1.92kSD 0.26 (63 lobes) and 1.96+SD 0.24 (73 lobes) times the initial weight of the lobe, respectively. Repeated lung washings caused a progressive increase in retractive forces of the lung as shown by a decrease in total lobe air volume (TLV) (fig. 1) and Vo/,,, (lung air volume at 10 cm H,O P,,/lung air volume at 30 cm Hz0 P,p x 100) (fig. 2). Air volume at 30 cm H,O per g initial lung weight (V,,) was 12.77fSD 3.06 ml for 8 lobes after 9th washing, a reduction of 10.6 % compared to the V_ of freshly excised lungs (VW, = 14.29+SD 2.24 ml for 32 lobes). When the volume of intraalveolar isotonic saline was added to the air volume, the ‘air+fluid’ volume at 30 cm H,O per g initial lung weight increased to 13.94+SD 3.16 ml for the above
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Fig. 1. Effect of repeated lung lavage on total lung air volume (TLV) : Solid lines represent the lobe air volume at 30 cm H,O Pip and broken lines represent the lobe weight. Closed circles are the data for a left lower lobe. Wash number 1 to 9 were done with isotonic saline (see Methods). TLV progressively decnasbd with each lavage with no significant change in lobe weight after wash + 1. Wash # 10 was done with the fluid collected from wash # 1 which contained high axtamtration of surfactant (tig. 3) in order to htcrease the concentration of surfactant in the lung at the end of lung lavage. This resulted in an increase in TLV. A subsequent lung lavage (+ 11) with isotonic mlme again reduced T’LV. Open circles are the data ‘for the right lower lobe (from the same animal) which was lavaged only once (# 1) with isotonic saline and repeated air inflations done without further lung lavage. The lobes were degamed prior to each air inflation (see Methods).
104
E. E. FARIDY
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Wash Number Fig. 2. Effect of repeated lung lavage on pressure-volume characteristics of excised lung. Closed circles represent the V%,, (air volume at 10 cm H,O P,,/air volume at 30 cm H,O P,r x 100) after each lung lavage in a left lower lobe. Wash number 1 to 9 were done with isotonic saline (see Methods). Wash # 10, was done with the fluid collected from wash # 1 which contained a high concentration of surfactant (fig. 3) in order to increase the con~ntration of surfactant in the lung at the end of lung lavage. This resulted in an increase in VA,,. A subsequent lung lavage (x.11) with isotonic saline again decreased the V’&,. Open circles represent the V%,, of the right lower lobe (from the same animal) which was lavaged only once (# 1) with isotonic saline and repeated PV curves were done without further lung lavage. The lobes were degassed prior to each PV curve (see Methods).
8 lobes. Table 1 shows the pressure-volume characteristics of the lungs after 9 washings. The tissue elastic retractive forces on the other hand were unchanged by repeated lung lavage when compared with the unwashed lungs. Table 2 shows the results of
TABLE 1 Lobe air volume at various static deflating pressures, expressed as percent lobe air volume at 30 cm H,O transpulmonary pressure Pf,
~~(32) (air vohrme)
After wash #9(S) (air volume)
After wash #k9(8) cair + saline’ volume)
20 15 10 7.5 5 2.5 0
94.35kO.18 88.08 *o&l 75.77 *0.90 64.72k1.17 50.38i1.15 30.65 kO.73 7.74kO.39
86&I&0.94* 75.94f 1.38* 52.04 f 2.60;
87.79 f0.83* 78.04f1.21* 56.16f2.21’
23.06 f 2.26*
29.65f2.11*
3.30*0.73*
11.56f1.07*
FE : freshly excised lobes of dogs lungs. Values are. given as mean f SE. Numbers in parentheses indicate the number of lobes studied. In calculating the data for the third column an assumption was made that the isotonic saline remaining in the lung after wash ~9 was in the alveoli and that it contributed in distending the lobe. Therefore a constant volume (that ofkotonic saline in the lung) waaadded to air volume at each Ptp and percentage of the total lobe volume (air + fluid) calculated. * Different from freshly excised lung (P c 0.001).
DISTENSION AND SURFACTANT
RELEA!B
105
TABLE 2 Lobe saline volume at different deflating pressures expressed as percent lobe saline volume at 12 cm H,O transpulmonary pressure
Ptp(c-mWV
Freshly excised (4)
After wash # 9(4)
11 10 9 8 7 6 5 4 3 2 1 0
9s.7*0.17 96.9 &0.40 94.OkO.78 89.7 f 1.33 83.9kl.91 76.4k2.49 66.9 f2.66 56.9 *2.26 46.8kl.76 37.lkl.21 28.4kO.74 18.7+0.60
99.OkO.11 97.3 +0.36 94.9 kO.52 90.8 +0.83 85.2kl.52 77.8kl.94 68.3k2.40 57.9 k2.28 47.2 f 1.75 37.4+ 1.16 28.5 +0.67 18.9kO.99
Values are means &SE. Numbers in parentheses indicate the number of lobes studied. The PV curve after wash # 9 is calculated by adding the volume of isotonic saline remaining in the lobe after wash #9 (lobe weight-initial lobe weight) to the volumes read from the burette for each deflating pressures (see Methods).
isotonic saline deflation pressure-volume measurements on 4 lower lobes when freshly excised and after 9 consecutive lavages. Figures 1 and 2, and table 3 indicate that increase in retractive forces of the lung following removal of surfactant by repeated washings was partially reversible when the concentration of surfactant of the fluid retained in the lung was increased. The quantity of surfactant in the lobe after wash # 9 was increased by means of: (a) washing the lobe with the fluid collected from wash 4 1 which contained high con-
TABLE 3 V%,, before and after instillation of surfactant in the excised lavaged lower lobes Instilled material
Before
After
Wash # 1
53.4 49.0 43.5 44.8 50.9 48.3 f 1.9
64.5 55.3 51.7 55.4 58.8 57.1 *j2.2*
DPL Mean fSE
V%,Cl = air volume at 10 cm H,O P&sir volume at 30 cm H,O P* x 100. Wash 9 1 = fluid collected after lavage # 1. The lobe was washed with this fluid atIer wash X 9. DPL = 2 mg DPL in 5 ml isotonic saline (see text). Note an increase in V%,, in each case. * Different from before.’ (P < 0.001).
106
E. E. FARIDY
centration of surfactant (fig. 3), or (b) instillation of 2 mg dipalmitoyl phosphatidyl choline in the lobe as follows: two mg DPL was dissolved in l-2 ml 2Propanol (isopropyl alcohol) and the latter poured on 5 ml isotonic saline in a beaker. After 2-Propanol had evaporated the saline was instilled in the lobe assuming that at least a portion of the DPL entered the alveolar spaces. The V%,, of these lobes before and after instillation of surfactant is shown in table 3. CHARACTERISTICS OF WASH FLUID
The 1st wash fluid was opalescent but subsequent washes progressively cleared. The wash fluids collected after the 9th and 10th lavages were 98.5fSD 6.7 (72 lobes) and 92.8 f SD 7.3 (72 lobes) percent of the volume of isotonic saline used for lavage, respectively. The minimum surface tension (y,,& of the lung washings (fig. 3) progressively increased with repeated washings and reached a value of 22.5 f SD 2.7 dyne/cm at the 9th washing for 55 lobes studied.
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Fig. 3. Minimum surface tension of the 1st to 9th lung washing (solid line). The bars represent 1 SD to either side of the mean. Numbers in parentheses represent number of lobes studied. The broken line is the lecithin concentration in wash fluid 1 to 9 obtained from one lower lobe.
The lecithin content of lung washings, conversely, decreased and in the 9th wash was 7.18 _+SD 2.49 mg/lOO g lung for 29 lobes studied. In fig. 3 is shown the lecithin content of wash # 1 to #9 which were obtained from only one lower lobe to point out the progressive reduction in the lecithin content of the lung washes. The lecithin as a percentage of the total phospholipids in wash # 9 was 69.9 f SD 5.1% for 13 lobes studied. The saturated lecithin as a percentage of the total lecithin was 66.2 f SD 4.9 % in wash # 9 measured from 3 lobes.
DISTENSION AND SURFACTANT RELEASE
107
EFFECT OF DISTENSION
effect of static inflation (constant stretch) at different transpulmonary pressures, on the surface activity of the washings from the lungs at the end of static inflation is shown in fig. 4. There is a direct relationship between the surface activity and the pressure at which the lung was kept statically inflated.
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Fig. 4. Relationship between minimum surface tension of wash # 10 (expressed as a percentage of wash + 9) obtained from lobes kept at constant inflation for 3 hr with air at room temperature, and inflation pressure. Each point represents data obtained from one lower lobe at the end of 3 hr. A regression line calculated by the least-squares technique had a slope of - 1.65kO.22 which was significantly different from zero (P < 0.001).
Figure 5 shows the effect of inflation time on the surface activity of lung washing. The yminof the 10th wash decreased as the inflation time of the lobe it was obtained from increased from zero time (i.e. one full inflation and deflation) to 3 hr constant inflation at room temperature. However, when wash # 10 was obtained from lobes after one full inflation and deflation and kept in a beaker at room temperature for up to 3 hr, the surface tension was not different from that measured at 0 time (fig. 5). Figure 6 shows the effect oftemperature of the~environment where the lobes were kept at constant inflation with air on the surface activity of lung kashing. For these experiments, the lobes were kept in a humidified box where the tam~rature was maintained at a constant level throughout the experiment. Prior to wash # 10, the lobe was kept inflated at room temperature for 15 minutes to allow the temperature of the lobe to equilibrate with room temperature (Faridy et al., 1966). The surface activity of lung washing from lobes kept at constant inflation with air at 6 “C did not change. Washings from lobes kept inflated at higher temperatures showed increased surface activity with increase in temperature. In fig. 7 is shown the effect of different concentrations of 0, in N,, used for
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Fig. 5. Minimum surface tension of lung wash # 10 (expressed as a percentage of wash #9) from lobes kept at constant inflation (at 30 cm H,O with air at room temperature) at different times. Time 0 indicates one full inflation and deflation (lasting about 1 min).
inflating the lungs at room temperature, on the surface activity of lung washing. The surface activity of lung washing increased as O2 concentration of inflating gas increased from zero to 20 ‘Aand decreased with further increase in 0, concentration by 100 % 0,. Figure 8 shows the lecithin content of wash 10 expressed as a percentage of wash 9 for different experimental conditions. An increase in lecithin content was only
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DISTENSION
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109
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Fig. 7. Minimum surface tension of wash # 10 (as a percentage of wash #9) from lobes kept at room temperature at a constant inflation of 30 cm H,O for 3 hr with gas containing different concentration of oxygen in nitrogen. The lobe was placed in a plexiglass box supplied with a continuous flow of humidified gas similar to that used for inflating the lobe.
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Fig. 8. Lecithin of lung wash # 10 (expressed as a percentage of wash #9) from different experimental conditions: A = The lobe was washed (# 10) immediately after wash #9. B = The lobe was kept at 0 cm H,O (RV), at room temperature for 3 hr. E = Constant inflation (CI) at 30 cm H,O with air, at 6 C for 3 hr. C = One full inflation with air to 30 cm H,O lasting about 1 min. D, F and G = Constant inflation at 30 cm H,O, for 3 hr, at room temperature, with air, N, and 0,, respectively.
110
E. E. FARIDY
noted in washings from lobes kept at constant inflation with air at room temperature for 3 hr. In four experiments the fully saturated lecithin was measured in wash # 10. The values of saturated lecithin as a percentage of the total lecithin for different conditions in fig. 8 were as follows: for lobe kept at RV (fig. 8B) = 66.0; for constant inflation for 3 hr with air (8D) = 71.7, with N, (8F3 = 62.8, and with 0, (8G) = 65.9. Figure 9 shows the relationship between lecithin content and ymin of wash 10 expressed as a percentage of wash 9. 1401 ? x 120. 0) ? & = 100 c 2
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Fig. 9. ReIationship between lecithin content and minims surface tension of wash # 10; expressed as a percentage of wash #9. Each point represents the mean *SE to either side of the mean. A regression line calculated by the least squares technique had a slope of -0.57kO.08 which was significantly different from zero (P c 0.01). Broken lines represent 2 SD to either side of the mean.
The purpose of repeated lung lavage was to reduce the surface activity of the wash fluid (fig. 3) by lowering the concentration of the surfactant. Small changes in the activity of subsequent washes which might result from the release of small quantities of surface active material could then be detected easily. Inspite of the fact that the alveolar structure of the lavaged lung remained unchanged, an observation similar to that of Brain and Frank (1968) who lavaged rat lungs, it differed from the freshly excised lung in that a great nwnber of cells were removed from the lung, and at the end of washing some fluid was trapped in the lung. In addition, repeated lung lavage increased the retractive forces of the lung (table 1). This was as a result of repeated removal of surfactant from lungs and not owing to
DISTENSION
AND SURFACI-ANT
RELEASE
111
the presence of intraalveolar fluid. Because of the smaller air volumes for a given transpulmonary pressure, smaller quantities of oxygen were available for the lung tissue. Being aware of these factors and the fact that 0, concentration of the intraalveolar air is continuously decreasing (Faridy a#d Naimark, 1971), one could conceive that the data from constant inflation with air at low transpulmonary pressures (5 and 10 cm H,O) in fig. 4 is mainly influenced by the smaller 0, content in the alveoli and not by the degree of distension of the tissue. To test this possibility, the following experiments were conducted in 4 lobes. Two lobes were kept at constant inflation with 40 % 0, in N, at pressures 5 and 10 cm H,O. The results were not different from those in fig. 4 which were inflated with air. Two other lobes were kept inflated at 5 and 10 cm H,O P,, with air for 3 hrs but every hour the lobes were slowly deflated and then inflated with air. This was done to increase the 0, concentration within the lung. The results were again not different from those in fig. 4. It appears, therefore, that the results in fig. 4 are mainly as a result of the degree of distension of the tissue. An increase in surface activity and lecithin content of wash 10 could result from either the secretion of surface active substance into alveoli or the expulsion of the same from ruptured cells. Cell counts from wash 9 and 10 indicated no significant change in cell numbers and no significant change in the number of ruptured cells. Therefore it appears more likely that the surface active substance has been released into the alveoli as a result of constant stretch. Whether this surfactant came from the total pool present in the lung tissue or was a new product could not be distinguished in this study. The amount of lecithin released in 3 hr at room temperature from lungs kept at constant inflation of 30 cm H,O P,, with air can be calculated from the following data. The average lecithin content in wash 9 and wash 10 for the five lobes in fig. 8D is 6.90 and 8.29 mg/lOO g lung, respectively. The average lecithin content in wash 10 (when washed immediately after wash 9).for the 4 lobes in fig. 8A is 63.8 % of that for wash 9. If no lecithin was released during constant inflation for 3 hr, the lecithin content of 5 lobes in fig. 8D should have been equal to 6.90 x 63.8/100 = 4.40 mg/ 100 g lung. Therefore the amount of lecithin released in 3 hr of constant inflation is 8.29-4.40 = 3.89 mg/lOO g lung. This calculation is based on the assumption that removal of surfactant from the alveoli does not occur during static inflation of the lung. If, however, surfactant is removed from the alveoli during this period and distension also accelerates surfactant removal, the amount of surfactant secreted would have been larger than that calculated. Since greater quantities of lecithin were collected from lung lavages after static inflation, then the rate of secretion must have been greater than the rate of removal of surfactant. Although fully saturated lecithin was measured only in few instances, the percentage of saturated to the total lecithin in wash 10 was comparable with that of wash 9. This suggests that the increase in lecithin content of wash 10 was as a result of an increase in both saturated and unsaturated lecithins.
112
E. E. FARIDY
The findings suggest that surfactant release by distension involves an active process. The evidence is from two sources. First, the process was markedly temperature dependent. Surfactant release did not occur when the lobe was kept in the cold, but increased as the temperature was raised. It is not in~n~ivable that some type of passive process could also be hmperature dependent; e.g. diffusion of.an essential substance from within the tissue to the surface. But when the effect of temperature is taken in conjunction with the finding that an absence of oxygen resulted in failure of release of surfactant, it would appear likely that an active process involving some function of viable cells was responsible for this phenomenon. It has been shown that lung slices and whole lungs in Y&O are metabolically active for at least 34 hr after excision from the animal in that they consume oxygen and exhibit glycolytic, lipolytic, and lipogenic activity (Barron et al., 1947; Evans et al., 1934; Felts, 1965 ; Heinemann, 1961; Levey and Gast, 1966). In a previous study we have shown that the rate of O2 consumption of excised lungs of dogs over a 3-hr period was only decreased by 3-8 % (Faridy and Naimark, 1971). In the present study the interval between exsanguination of the animal and the end of experiments did not exceed 4 hr. In a previous study Faridy and Naimark (1971) showed that the (iol of statically inflated lobes was directly dependent on the degree of inflation. Because of similarities between the effects of distension on lung metabolism and the ‘stretch response’ phenomenon observed in the striated and smooth muscles it was postulated that the smooth muscle component may be the element primarily affected by mechanical deformation of the lung. The present study suggests that the metabolism of cells other than smooth muscle is also affected by distension. Since the process of surfactant release is oxygen dependent one could conclude that it contributes to the rise in ci,, in response to distension. The mechanism by which mechanical deformation (distension) exerts its metabolic effect in causing release of surfactant is unknown. A variety of possibilities exist including activation of membrane-bound enzymes by stretch of cell walls and subcellular membranous structures and changes in ionic permeability of membranes with subsequent stimulation of metabolism. The present findings confirm the previous notion (Faridy et al., 1966; Faridy, 1973) that recovery of the pressure-volume curve from the effects of ventilation following constant inflation in a metabolically active lung, is achieved by release of surfactant into the alveoli and that replenishment of surfactant is enhanced by distension of the lung. Whether distension has a similar effect on release of surfactant in a non-washed lung and at 37 “C has yet to be defined. An interesting finding is the inhibition of surfactant release by pure oxygen, which may be a contributing factor in the development of symptoms of oxygen toxicity in the lung. Our previous studies (Faridy et al., 1966) indicate that when excised lungs are ventilated with air for 3 hr, the retractive forces of the lung increase. These effects of ventilation are fully reversed when the lungs are kept at constant inflation with air for 3 hr. In the present study (table 4) freshly excised lobes ventilated with air (VT = 30 ‘A TLV; f = 12/min; EEP = 0 cm H,O) for 3 hr at room temperature,
113
DISTENSION AND SURFACTANT RELEASE
did not r~ver~omplete~y when sub~quently kept at constant inflation of 30 cm H,O pressure with pure 0, for 3 hr. TABLE 4 Recovery from the effects of ventilation after static inflation with oxygen .-. -
P,p (cm I-W)
Control (5)
Vent. (air) (5)*
Vent. (air)+CI
30 20 15 10 5 0
98.2 +0.22 93.6kO.65 85.3k2.18 68.6 +4.06 44.4+ 3.54 8.2kO.32 __--.
96.5 kO.66’ 88.3fl.91’ 78.0+3.01+ 52.4i4.90’ 30.453.22’ 6.1 f0.91”
97.81tO.31’ 91.4+ 1.12” 81.9,2.35+ 61.2k3.86’ 39.5 +2.45++ 8.7+0.33 --
(0,) (S)**
--..-
Values are means + SE and indicate lobe air volume at different deflating pressures expressed as percent lobe air volume at 40 cm H,O transpulmonaty pressure. The deflation pressure-volume curves were obtained in lobes when freshly excised (control), after ventilation with air, and linaliy after same lobes were kept at constant inflation (CI) with pure oxygen. The lobes were always degassed prior to PV measurements (see Methods). Numbers in parentheses indicate the number of lobes studied. * Ventilated with air (VT = 30‘A of TLV; f = lymin; EEP = 0 cm H,O; duration = 3 hr). ** Following ventilation (as indicated above), the lobes were kept at constant inflation (P,, 30 cm H,O) with 100 od 0, for 3 hr. The lobe was also surrounded with 0,. ’ Different from controls (P < 0.05 - < 0.01). ‘+ Different from control (P < 0.10). Comparison of differences was made by means of paired r-test.
Acknowledgements The author expresses his deep appreciation to Dr. J. Thliveris for the EM studies
and to Adolph Wellemin, Walfried Jansen and Kwok-Tung Ghan for their excellent technical assistanee.
References Barron, E. S. G., Z. Miller and G. R. Bartlett (1947). The metabolism of lung as determined by a study of slices and ground tissue. J. Biol. C/rem. 171: 791-800. Brain, J. D. and N. R. Frank (1968). Recovery of free cells from rat lungs by repeated washings. J. Appl. Physiol. 25: 63-69.
Bra&e, G. (1949). Studies on lipids in the nervous system : total phosphorus determination. Rcta Physiol. &and. 63: 39-40.
Evans, C. L., F. Y. Hsu and T. Kosaka (1934). Utilization of blood sugar and formation of lactic acid by the lungs. J. Physiol. (Land.) 82: 41-61. Faridy, E. E., S. Permutt and R. L. Riley (1966). Effect of ventilation on surface forces in excised dogs’ lungs. J. Appl. Physiol. 21: 1453-1462. Faridy, E. E. and A. Naimark (1971). Effect of distension on metabolism of excised dog lung. J. Appf. Physd. 31: 31-37.
114
E. E. FARIDY
Faridy, E. E. (1973). Effect of hydration and dehydration
on elastic behavior of excised dogs’ lungs.
J. Appl. Physiol. 34: 597-605.
Felts, J. M. (1965). Carbohydrate and lipid metabolism of lung tissue in vitro. Med. Thoruc. 22: 89-99. Fiske, C. H. and Y. Subbarow (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66: 375400.
Folch, J., M. Lees and G. H. Sloane-Stanley (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. Greenfield, L. J. and G. 0. -Kimmell (1967). Application of pneumatic techniques to surface tension determinations. The surfactometer. J. Surg. Res. 7: 276-297. Gribetz, I., N. R. Frank and M. E. Avery (1959). Static volume-pressure relations of excised lungs of infants with hyaline membrane disease, newborn and stillborn infants. J. Clin. Inuesr. 38: 21682175. Heinemann, H. 0. (1961). Free fatty acid production by rabbit lung tissue in vitro. Am. J. Physiol. 201: 607610.
Levey, S. and R. Gast (1966). Isolated perfused rat lung preparation. J. Appl. Physiol. 21: 313-316. Luft, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409414.
Mangold, H. K. (1961). Thin-layer chromatography of lipids. J. Oil Chem. Sot. 38: 708-727. Parker, F. and N. F. Peterson (1965). Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6: 455-460. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Ceil Biol. 17: 208212. Richardson, K. C., L. Jarrett and E. Finke (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35 : 3 13-323. Sabatini, D. D., K. Bensch and R. J. Barnett (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17: 19-58.
North-Holland Publishing Company, Amsterdam
EFFECT OF DISTENSION ON RELEASE OF SURFACMNT DOGS’ LUNGS’ ’
IN EXCISED
EDMUND E. FARIDY Department of Physiology, University of Manitoba, Winnipeg, Canada
Abstract. The freshly excised lower lobes of lungs of dogs were washed by filling and emptying the lobe with isotonic saline. The wash fluid was then discarded and the procedure was repeated 9 times. The 9th wash was collected and used for measurements of surface tension and lecithin content. The lobe was then constantly inflated for 3 hr; washed again with isotonic saline (10th wash) and surface tension and lecithin content of wash fluid measured. When the lobe was inflated with air, at room temperature, the surface activity and the lecithin content of the 10th wash were increased in comparison to tbe 9th wash. This increase was not noted when the lobe was inflated with 100 % N, or 100 y0 O2 at room temperature, or with air at 6” C. Increase in the surface activity of the 10th wash was directly related to the inflating pressures used. Thii study suggests that distension of the lung enhances the release of surfactant and that this &ase is a metabolically active process. It appears also that 100% 0, has an inhibitory effect on this pr-s. Lung distension, effect on surfactant Lung lavage Lung metabolism
Oxygen toxicity Pressure-volume Surface tension
measurements, factors afkctmg
In freshly excised lungs, ventilation depletes surfactant, resulting in an increase in lung retractive forces (Faridy et al., 1966). The effects of ventilation are prevented by the application of an end-expiratory pressure and are reversed if the lungs are kept at a constant volume for several hours, provided the temperature of the environment is appropriate and the lungs are not deprived of oxygen at any stage of the experiment. In a metabolically inactive lung (such as dehydrated-rehydrated lung) (Faridy, 1973) the rate of depletion of surfactant by ventilation is greatly increased; positive end-expiratory pressure does not prevent depletion of surfactant ; and the effect of ventilation is not reversed by constant inflation. These observations led us to hypothesize (Faridy et al., 1966; Faridy, 1973)that recovery of the pressur+volume curve in a metabolically active lung occurs as a Accepted for publication 9 March 1976.
’ Preliminary reports of this study have been presented at the meeting of the American Physiological Society: Fed. Proc. (1973) 32: 401. ’ This study was supported by the Medical Research Council of Canada. 99
100
E. E,
FARIDY
result of newly formed surfactant replacing that depleted by ventilation, and that distension enhances the replenishment of surfactant. The present experiments were designed to test this hypothesis.
Methods Seventy-three mongrel dogs of either sex, free of respiratory infections, weighing 7.4-12.8 kg, were used for this study. The animals were anesthetized with sodium pentobarbital (30 mg/kg body weight), heparinized and exsanguinated. The lungs were excised; the left and the right lower lobes were separated, and weighed. A plastic cannula was tied into the main bronchus of each lobe, and the pulmonary vessels were ligated. The lobes were then degassed in a vacuum jar, as previously described (Faridy, 1973) and lavaged 9 times. Each lung lavage consisted of filling and emptying the lobe with isotonic saline (5 ml/g initial lobe weight) 4 times from and into a syringe. For the first 2 filling and emptying, the lobe was placed on its mediasnnal surface and for the latter 2 filling and emptying it was placed on its thoracic surface. The wash fluid was then discarded and the procedure repeated 9 times using fresh saline each time. The lobe was weighed prior to the 9th lavage. The fluid from the 9th lavage was collected. The interval between ex~n~n~tion of the dog and the termination of lung ‘lavage #9 was about 60 min. The lobe was then kept at residual volume or at static inflation at predetermined transpulmonary pressures which ranged from 5 to 30 qn H,O. This was accomplished by connecting the lobe to a T tube, one limb of which was kept under water and the other connected to an air tank. The lobe was fully inflated to 30 cm H,O pressure by lowering the limb of the T tube 30 cm under water, then deflated to a predetermined pressure by raising the limb of the T tube to the predetermined depth under water. The lobe was kept constantly inflated by the continuous ,airtlow. The l&es were kept at a highly humidified atmosphere (Mistogen tent) to prevent dehydration. At the end of 3 hr the lobe was weighed and lavaged once again, as described above, with isotonic saline (10th wash). The lobe was not degassed prior to the 10th wash in order to prevent spilling the isotonic saline retained in the lobe after wash 99 As seen in table 1, the volume of air retained in the lung at 0 cm H,O pressure after tish =#9 was small and could have little effect on the distribution of wash fluid in the lung. In addition, most of the trapped air was withdrawn into the syringe during the first and second filling and emptying maneuvers of wash # 10. Fluids from the 9th and 10th washes were collected and used for measurements of surface tension and lecithin content. The results of the 10th wash were expressed as a percentage of the 9th wash to facilitate comparison between experiments. If, during the lung lavage, there was a leak in the lobe or the wash fluid was not clear but light red or red in color (~n~~a~ with blood), such lobes and lung washes were excluded from the study.
DISTENSION
AND SURFACTANT
RELEASE
101
SURFACE TENSION
wash fluid was stirred for 5 minutes by means of a magnetic stirrer, and a 40 ml sample was .&ken. Surface tension was then measured on this sample at room temperature, on a modified Wilhelmy surface tension balance (Kimray-Greenfield surfactometer) (Greentield and Kimmell, 1967) after continuous cyclic compression and expansion of the surface area (between 15 and 100 %) for 2 hr, each cycle lasting 3 min. The lowest surface tension recorded was designated as the minimum surface tension (Y,,,~,,)of each lung washing. In experiments where lung lavage washings were used for surface tension measurements, the lobe after wash 9 wasemptied of isotonic saline until the weight of the lobe was within 1 g of that prior to the 9th washing.
The
AIR DEFLATION PRESSUR-VOLUME
CURVE
The cannulated degassed lobes were attached to the T tube of a pressure-volume apparatus similar to that previously described by Gribetz et al. (1959). The lobe was then inflated with air to 30 cm H,O pressure. This inflation pressure was maintained until the lobe air volume remained constant for 10 sec. The air volume observed at this transpulmonary pressure, considered as total lung air volume (TLV), was designated as 100 % and each volume subsequently observed after deflation to a predetermined transpulmonary pressure (20, 15, 10,5, and 0 cm H,O) was expressed as a percentage of TLV. These pressures were maintained until the volumes were stable for 15 sec. If, during the procedure, the lung air volume did not remain constant at high pressures, air leaks were assumed to be present, and such lungs were excluded from the study. All pressure-volume measurements were performed at room temperature. ISOTONIC SALINE DEFLATION PRES!WRl-VOLUME
CURVE
The cannulated degassed lobe was mounted in an isotonic saline bath, and its bronchus was attached to the T tube of a saline pressure-volume apparatus. One limb of the T tube was connected to a saline-filled burette. The other limb was connected to a vertical tube placed next to the saline bath. The vertical tube, containing saline at the same level as that of the burette, together with a second vertical tube connected to the bath, served as a monometer. By raising and lowering the burette, saline was displaced to and from the attached lo& at pressures simultaneously registered by the manometer. The lobe was inflated with isotonic saline to 12 cm H,O pressure. After stability of the volume at this inflation pressure was achieved, a deflation pressur~volume curve was obtained by lowering the(inflation pressure at decrements of 1 cm II,0 at intervals of 2 min. The saline volume observed at 12 cm H,O transpulmonary pressure, considered as total lung saline volume (TLV), was designated as 100 %, and each volume subsequently observed after deflation to’s predetermined transpulmonary pressure was expressed as a percentage of TLV.
IO2
E. E. FARIDY
If, during the procedure, the lung saline volume did not remain constant at high pressures, or the lobe weight at 0 cm H,O Ptp exceeded the initial lobe weight plus the weight of the saline recorded, saline leaks were assumed to be present, and such lungs were excluded from the study. All press~e-vol~e m~sur~ents were performed at room temperature. LECITHIN
The lung washing was ~ntrifug~ at I650 G for 20 minutes and cell free supematant was then spun at 30,000 x g for 1 hr. The lipids were extracted, from the spun sediment by chloroform-methanol (2 : 1) and washed according to the method of Folch it al. (1957). The sample was dried in a water bath at 40-50 “C under nitrogen and the dried extract reconstituted to 0.5 ml with chloroform. An aliquot of the lipid extract was plated on an activated silica gel-H plate and the lipid fractions separated using a solvent system containing c~orofo~-me~anol-attic acid-water (25 : 15 : 4 : 2) (Parker and Peterson, 1965). The plate was then exposed to iodine vapor and the lecithin spot was identified. After the iodine vapor had evaporated from the plate, the lecithin spot was aspirated into a test tube, the fully saturated lecithin fraction was isolated by mercuric acetate adduction (Mangold, 1961), and the lipid phosphor determined by Brante’s rn~~~tion (Brante, 1949)of the method of Fiske and Subbarow (1925). For calculation of lecithin content in the wash fluid two assumptions were made, namely, that the fluid remaining in the lung prior to the 9th and 10th lavages was in the alveoli and that at the end of lavage it had the same concentration of lecithin as that of the collected fluid. Knowing the inundation of lecithin in the collected fluid, and the amount of fluid retained in the lung (lobe wei~t-~itial lobe weight), the total lecithin content was then calculated. ELECTRON MICR~PIC
STUDIES
Samples of lung tissue were immersed in cold fixative (6.25 % ~u~dehyde in 0.1 M cacodylate buffer, pH 7.4), Sub~~ntly, the lung tissues were transferred into vials containing 6.25 % glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 2 hours at 4 “C. The tissues were rinsed for 24 hours at 4 “C in 0.1 M cacodylate buffer (pH 7.4) containing 0.2 M sucrose (Sabatini et al., 1963). They were then postfixed for 4 hours at 4 “C in 2 % osmium tetroxide buffered in 0.1 M cacodylate (pH 7.4) containing 0.2 M sucrose. Following rapid dehydration, the tissues were embedded in Epon 812 (Luft, 1961).Thick 1 pm sections were stained by R&a&on’s tech& que (Richardson et al., 1960), and examined for general orientation with the light microscope. Thin sections were mounted for electron microscopy on naked 300-mesh copper grids, stained with uranyl aoetate (Luft, 1961) and lead citrate (Reynolds, 1963)and photographed in a RCA EMV 3F electron microscope. A r-test of paired and unpaired variates was empioyed to ascertain differences among the means of two groups of observations.
103
DISTENSION AND SURFACIANT RELEASE
CHARACl’ERIST’ICSOF LAVAGED LUNG
After 9 lung washings the alveolar structure remained unchanged. The distinction between the washed and unwashed lungs was nearly impossible in both light and electron mi~o~pic studies. This was also true in lavaged lobes that had been statically inflated with air for 3 hr and washed for the 10th time, A portion of isotonic saline was trapped in the lung at the end of lavage. The lobe weights, therefore, prior to the 9th and 10th washes were greater by 1.92kSD 0.26 (63 lobes) and 1.96+SD 0.24 (73 lobes) times the initial weight of the lobe, respectively. Repeated lung washings caused a progressive increase in retractive forces of the lung as shown by a decrease in total lobe air volume (TLV) (fig. 1) and Vo/,,, (lung air volume at 10 cm H,O P,,/lung air volume at 30 cm Hz0 P,p x 100) (fig. 2). Air volume at 30 cm H,O per g initial lung weight (V,,) was 12.77fSD 3.06 ml for 8 lobes after 9th washing, a reduction of 10.6 % compared to the V_ of freshly excised lungs (VW, = 14.29+SD 2.24 ml for 32 lobes). When the volume of intraalveolar isotonic saline was added to the air volume, the ‘air+fluid’ volume at 30 cm H,O per g initial lung weight increased to 13.94+SD 3.16 ml for the above
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Fig. 1. Effect of repeated lung lavage on total lung air volume (TLV) : Solid lines represent the lobe air volume at 30 cm H,O Pip and broken lines represent the lobe weight. Closed circles are the data for a left lower lobe. Wash number 1 to 9 were done with isotonic saline (see Methods). TLV progressively decnasbd with each lavage with no significant change in lobe weight after wash + 1. Wash # 10 was done with the fluid collected from wash # 1 which contained high axtamtration of surfactant (tig. 3) in order to htcrease the concentration of surfactant in the lung at the end of lung lavage. This resulted in an increase in TLV. A subsequent lung lavage (+ 11) with isotonic mlme again reduced T’LV. Open circles are the data ‘for the right lower lobe (from the same animal) which was lavaged only once (# 1) with isotonic saline and repeated air inflations done without further lung lavage. The lobes were degamed prior to each air inflation (see Methods).
104
E. E. FARIDY
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Wash Number Fig. 2. Effect of repeated lung lavage on pressure-volume characteristics of excised lung. Closed circles represent the V%,, (air volume at 10 cm H,O P,,/air volume at 30 cm H,O P,r x 100) after each lung lavage in a left lower lobe. Wash number 1 to 9 were done with isotonic saline (see Methods). Wash # 10, was done with the fluid collected from wash # 1 which contained a high concentration of surfactant (fig. 3) in order to increase the con~ntration of surfactant in the lung at the end of lung lavage. This resulted in an increase in VA,,. A subsequent lung lavage (x.11) with isotonic saline again decreased the V’&,. Open circles represent the V%,, of the right lower lobe (from the same animal) which was lavaged only once (# 1) with isotonic saline and repeated PV curves were done without further lung lavage. The lobes were degassed prior to each PV curve (see Methods).
8 lobes. Table 1 shows the pressure-volume characteristics of the lungs after 9 washings. The tissue elastic retractive forces on the other hand were unchanged by repeated lung lavage when compared with the unwashed lungs. Table 2 shows the results of
TABLE 1 Lobe air volume at various static deflating pressures, expressed as percent lobe air volume at 30 cm H,O transpulmonary pressure Pf,
~~(32) (air vohrme)
After wash #9(S) (air volume)
After wash #k9(8) cair + saline’ volume)
20 15 10 7.5 5 2.5 0
94.35kO.18 88.08 *o&l 75.77 *0.90 64.72k1.17 50.38i1.15 30.65 kO.73 7.74kO.39
86&I&0.94* 75.94f 1.38* 52.04 f 2.60;
87.79 f0.83* 78.04f1.21* 56.16f2.21’
23.06 f 2.26*
29.65f2.11*
3.30*0.73*
11.56f1.07*
FE : freshly excised lobes of dogs lungs. Values are. given as mean f SE. Numbers in parentheses indicate the number of lobes studied. In calculating the data for the third column an assumption was made that the isotonic saline remaining in the lung after wash ~9 was in the alveoli and that it contributed in distending the lobe. Therefore a constant volume (that ofkotonic saline in the lung) waaadded to air volume at each Ptp and percentage of the total lobe volume (air + fluid) calculated. * Different from freshly excised lung (P c 0.001).
DISTENSION AND SURFACTANT
RELEA!B
105
TABLE 2 Lobe saline volume at different deflating pressures expressed as percent lobe saline volume at 12 cm H,O transpulmonary pressure
Ptp(c-mWV
Freshly excised (4)
After wash # 9(4)
11 10 9 8 7 6 5 4 3 2 1 0
9s.7*0.17 96.9 &0.40 94.OkO.78 89.7 f 1.33 83.9kl.91 76.4k2.49 66.9 f2.66 56.9 *2.26 46.8kl.76 37.lkl.21 28.4kO.74 18.7+0.60
99.OkO.11 97.3 +0.36 94.9 kO.52 90.8 +0.83 85.2kl.52 77.8kl.94 68.3k2.40 57.9 k2.28 47.2 f 1.75 37.4+ 1.16 28.5 +0.67 18.9kO.99
Values are means &SE. Numbers in parentheses indicate the number of lobes studied. The PV curve after wash # 9 is calculated by adding the volume of isotonic saline remaining in the lobe after wash #9 (lobe weight-initial lobe weight) to the volumes read from the burette for each deflating pressures (see Methods).
isotonic saline deflation pressure-volume measurements on 4 lower lobes when freshly excised and after 9 consecutive lavages. Figures 1 and 2, and table 3 indicate that increase in retractive forces of the lung following removal of surfactant by repeated washings was partially reversible when the concentration of surfactant of the fluid retained in the lung was increased. The quantity of surfactant in the lobe after wash # 9 was increased by means of: (a) washing the lobe with the fluid collected from wash 4 1 which contained high con-
TABLE 3 V%,, before and after instillation of surfactant in the excised lavaged lower lobes Instilled material
Before
After
Wash # 1
53.4 49.0 43.5 44.8 50.9 48.3 f 1.9
64.5 55.3 51.7 55.4 58.8 57.1 *j2.2*
DPL Mean fSE
V%,Cl = air volume at 10 cm H,O P&sir volume at 30 cm H,O P* x 100. Wash 9 1 = fluid collected after lavage # 1. The lobe was washed with this fluid atIer wash X 9. DPL = 2 mg DPL in 5 ml isotonic saline (see text). Note an increase in V%,, in each case. * Different from before.’ (P < 0.001).
106
E. E. FARIDY
centration of surfactant (fig. 3), or (b) instillation of 2 mg dipalmitoyl phosphatidyl choline in the lobe as follows: two mg DPL was dissolved in l-2 ml 2Propanol (isopropyl alcohol) and the latter poured on 5 ml isotonic saline in a beaker. After 2-Propanol had evaporated the saline was instilled in the lobe assuming that at least a portion of the DPL entered the alveolar spaces. The V%,, of these lobes before and after instillation of surfactant is shown in table 3. CHARACTERISTICS OF WASH FLUID
The 1st wash fluid was opalescent but subsequent washes progressively cleared. The wash fluids collected after the 9th and 10th lavages were 98.5fSD 6.7 (72 lobes) and 92.8 f SD 7.3 (72 lobes) percent of the volume of isotonic saline used for lavage, respectively. The minimum surface tension (y,,& of the lung washings (fig. 3) progressively increased with repeated washings and reached a value of 22.5 f SD 2.7 dyne/cm at the 9th washing for 55 lobes studied.
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The lecithin content of lung washings, conversely, decreased and in the 9th wash was 7.18 _+SD 2.49 mg/lOO g lung for 29 lobes studied. In fig. 3 is shown the lecithin content of wash # 1 to #9 which were obtained from only one lower lobe to point out the progressive reduction in the lecithin content of the lung washes. The lecithin as a percentage of the total phospholipids in wash # 9 was 69.9 f SD 5.1% for 13 lobes studied. The saturated lecithin as a percentage of the total lecithin was 66.2 f SD 4.9 % in wash # 9 measured from 3 lobes.
DISTENSION AND SURFACTANT RELEASE
107
EFFECT OF DISTENSION
effect of static inflation (constant stretch) at different transpulmonary pressures, on the surface activity of the washings from the lungs at the end of static inflation is shown in fig. 4. There is a direct relationship between the surface activity and the pressure at which the lung was kept statically inflated.
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Fig. 4. Relationship between minimum surface tension of wash # 10 (expressed as a percentage of wash + 9) obtained from lobes kept at constant inflation for 3 hr with air at room temperature, and inflation pressure. Each point represents data obtained from one lower lobe at the end of 3 hr. A regression line calculated by the least-squares technique had a slope of - 1.65kO.22 which was significantly different from zero (P < 0.001).
Figure 5 shows the effect of inflation time on the surface activity of lung washing. The yminof the 10th wash decreased as the inflation time of the lobe it was obtained from increased from zero time (i.e. one full inflation and deflation) to 3 hr constant inflation at room temperature. However, when wash # 10 was obtained from lobes after one full inflation and deflation and kept in a beaker at room temperature for up to 3 hr, the surface tension was not different from that measured at 0 time (fig. 5). Figure 6 shows the effect oftemperature of the~environment where the lobes were kept at constant inflation with air on the surface activity of lung kashing. For these experiments, the lobes were kept in a humidified box where the tam~rature was maintained at a constant level throughout the experiment. Prior to wash # 10, the lobe was kept inflated at room temperature for 15 minutes to allow the temperature of the lobe to equilibrate with room temperature (Faridy et al., 1966). The surface activity of lung washing from lobes kept at constant inflation with air at 6 “C did not change. Washings from lobes kept inflated at higher temperatures showed increased surface activity with increase in temperature. In fig. 7 is shown the effect of different concentrations of 0, in N,, used for
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Time (minute)
Fig. 5. Minimum surface tension of lung wash # 10 (expressed as a percentage of wash #9) from lobes kept at constant inflation (at 30 cm H,O with air at room temperature) at different times. Time 0 indicates one full inflation and deflation (lasting about 1 min).
inflating the lungs at room temperature, on the surface activity of lung washing. The surface activity of lung washing increased as O2 concentration of inflating gas increased from zero to 20 ‘Aand decreased with further increase in 0, concentration by 100 % 0,. Figure 8 shows the lecithin content of wash 10 expressed as a percentage of wash 9 for different experimental conditions. An increase in lecithin content was only
0
0
..““‘..‘.( 4
8
12
18
20
24
Tompomtum(C’) Fig. 6. Minimum surface tension of lung wash + 10 (expressed as a pcrccntage ofwash +9) from lobes kept tit constant inflation for 3 hrs at 30 cm H,O with air at different tempmatures.
DISTENSION
AND SURFACTANT
109
RELEASE
120, i z $
loo-
t
60-
. :
l
. .
: .
. .
. .
.
s j P
60.
: f
40-
> .
.
.; P
20-
.
.
0
-0
5
10
20
20,
40
100
in N2
Fig. 7. Minimum surface tension of wash # 10 (as a percentage of wash #9) from lobes kept at room temperature at a constant inflation of 30 cm H,O for 3 hr with gas containing different concentration of oxygen in nitrogen. The lobe was placed in a plexiglass box supplied with a continuous flow of humidified gas similar to that used for inflating the lobe.
. t . . -_ ~~~~~~~~~~~~_~_~_ . . r 3
80.
P 3 6
80.
I .z
40
.
. 5
. . .
. .
. .
.
:
t
0
a
.
:
. ---_ . . .
20
A
B
0 min. 180 RV
C
D
0 min.180 C. Infl.
, Air
E
F
G
6°C I
N2
02
C.lnfl.
Fig. 8. Lecithin of lung wash # 10 (expressed as a percentage of wash #9) from different experimental conditions: A = The lobe was washed (# 10) immediately after wash #9. B = The lobe was kept at 0 cm H,O (RV), at room temperature for 3 hr. E = Constant inflation (CI) at 30 cm H,O with air, at 6 C for 3 hr. C = One full inflation with air to 30 cm H,O lasting about 1 min. D, F and G = Constant inflation at 30 cm H,O, for 3 hr, at room temperature, with air, N, and 0,, respectively.
110
E. E. FARIDY
noted in washings from lobes kept at constant inflation with air at room temperature for 3 hr. In four experiments the fully saturated lecithin was measured in wash # 10. The values of saturated lecithin as a percentage of the total lecithin for different conditions in fig. 8 were as follows: for lobe kept at RV (fig. 8B) = 66.0; for constant inflation for 3 hr with air (8D) = 71.7, with N, (8F3 = 62.8, and with 0, (8G) = 65.9. Figure 9 shows the relationship between lecithin content and ymin of wash 10 expressed as a percentage of wash 9. 1401 ? x 120. 0) ? & = 100 c 2
80.
P 3
60.
$ f 40 .Z B” -I t 201 20
40
60
so
100
bl min. of Lung Wash (#lo/#9
120
, 140
x 100)
Fig. 9. ReIationship between lecithin content and minims surface tension of wash # 10; expressed as a percentage of wash #9. Each point represents the mean *SE to either side of the mean. A regression line calculated by the least squares technique had a slope of -0.57kO.08 which was significantly different from zero (P c 0.01). Broken lines represent 2 SD to either side of the mean.
The purpose of repeated lung lavage was to reduce the surface activity of the wash fluid (fig. 3) by lowering the concentration of the surfactant. Small changes in the activity of subsequent washes which might result from the release of small quantities of surface active material could then be detected easily. Inspite of the fact that the alveolar structure of the lavaged lung remained unchanged, an observation similar to that of Brain and Frank (1968) who lavaged rat lungs, it differed from the freshly excised lung in that a great nwnber of cells were removed from the lung, and at the end of washing some fluid was trapped in the lung. In addition, repeated lung lavage increased the retractive forces of the lung (table 1). This was as a result of repeated removal of surfactant from lungs and not owing to
DISTENSION
AND SURFACI-ANT
RELEASE
111
the presence of intraalveolar fluid. Because of the smaller air volumes for a given transpulmonary pressure, smaller quantities of oxygen were available for the lung tissue. Being aware of these factors and the fact that 0, concentration of the intraalveolar air is continuously decreasing (Faridy a#d Naimark, 1971), one could conceive that the data from constant inflation with air at low transpulmonary pressures (5 and 10 cm H,O) in fig. 4 is mainly influenced by the smaller 0, content in the alveoli and not by the degree of distension of the tissue. To test this possibility, the following experiments were conducted in 4 lobes. Two lobes were kept at constant inflation with 40 % 0, in N, at pressures 5 and 10 cm H,O. The results were not different from those in fig. 4 which were inflated with air. Two other lobes were kept inflated at 5 and 10 cm H,O P,, with air for 3 hrs but every hour the lobes were slowly deflated and then inflated with air. This was done to increase the 0, concentration within the lung. The results were again not different from those in fig. 4. It appears, therefore, that the results in fig. 4 are mainly as a result of the degree of distension of the tissue. An increase in surface activity and lecithin content of wash 10 could result from either the secretion of surface active substance into alveoli or the expulsion of the same from ruptured cells. Cell counts from wash 9 and 10 indicated no significant change in cell numbers and no significant change in the number of ruptured cells. Therefore it appears more likely that the surface active substance has been released into the alveoli as a result of constant stretch. Whether this surfactant came from the total pool present in the lung tissue or was a new product could not be distinguished in this study. The amount of lecithin released in 3 hr at room temperature from lungs kept at constant inflation of 30 cm H,O P,, with air can be calculated from the following data. The average lecithin content in wash 9 and wash 10 for the five lobes in fig. 8D is 6.90 and 8.29 mg/lOO g lung, respectively. The average lecithin content in wash 10 (when washed immediately after wash 9).for the 4 lobes in fig. 8A is 63.8 % of that for wash 9. If no lecithin was released during constant inflation for 3 hr, the lecithin content of 5 lobes in fig. 8D should have been equal to 6.90 x 63.8/100 = 4.40 mg/ 100 g lung. Therefore the amount of lecithin released in 3 hr of constant inflation is 8.29-4.40 = 3.89 mg/lOO g lung. This calculation is based on the assumption that removal of surfactant from the alveoli does not occur during static inflation of the lung. If, however, surfactant is removed from the alveoli during this period and distension also accelerates surfactant removal, the amount of surfactant secreted would have been larger than that calculated. Since greater quantities of lecithin were collected from lung lavages after static inflation, then the rate of secretion must have been greater than the rate of removal of surfactant. Although fully saturated lecithin was measured only in few instances, the percentage of saturated to the total lecithin in wash 10 was comparable with that of wash 9. This suggests that the increase in lecithin content of wash 10 was as a result of an increase in both saturated and unsaturated lecithins.
112
E. E. FARIDY
The findings suggest that surfactant release by distension involves an active process. The evidence is from two sources. First, the process was markedly temperature dependent. Surfactant release did not occur when the lobe was kept in the cold, but increased as the temperature was raised. It is not in~n~ivable that some type of passive process could also be hmperature dependent; e.g. diffusion of.an essential substance from within the tissue to the surface. But when the effect of temperature is taken in conjunction with the finding that an absence of oxygen resulted in failure of release of surfactant, it would appear likely that an active process involving some function of viable cells was responsible for this phenomenon. It has been shown that lung slices and whole lungs in Y&O are metabolically active for at least 34 hr after excision from the animal in that they consume oxygen and exhibit glycolytic, lipolytic, and lipogenic activity (Barron et al., 1947; Evans et al., 1934; Felts, 1965 ; Heinemann, 1961; Levey and Gast, 1966). In a previous study we have shown that the rate of O2 consumption of excised lungs of dogs over a 3-hr period was only decreased by 3-8 % (Faridy and Naimark, 1971). In the present study the interval between exsanguination of the animal and the end of experiments did not exceed 4 hr. In a previous study Faridy and Naimark (1971) showed that the (iol of statically inflated lobes was directly dependent on the degree of inflation. Because of similarities between the effects of distension on lung metabolism and the ‘stretch response’ phenomenon observed in the striated and smooth muscles it was postulated that the smooth muscle component may be the element primarily affected by mechanical deformation of the lung. The present study suggests that the metabolism of cells other than smooth muscle is also affected by distension. Since the process of surfactant release is oxygen dependent one could conclude that it contributes to the rise in ci,, in response to distension. The mechanism by which mechanical deformation (distension) exerts its metabolic effect in causing release of surfactant is unknown. A variety of possibilities exist including activation of membrane-bound enzymes by stretch of cell walls and subcellular membranous structures and changes in ionic permeability of membranes with subsequent stimulation of metabolism. The present findings confirm the previous notion (Faridy et al., 1966; Faridy, 1973) that recovery of the pressure-volume curve from the effects of ventilation following constant inflation in a metabolically active lung, is achieved by release of surfactant into the alveoli and that replenishment of surfactant is enhanced by distension of the lung. Whether distension has a similar effect on release of surfactant in a non-washed lung and at 37 “C has yet to be defined. An interesting finding is the inhibition of surfactant release by pure oxygen, which may be a contributing factor in the development of symptoms of oxygen toxicity in the lung. Our previous studies (Faridy et al., 1966) indicate that when excised lungs are ventilated with air for 3 hr, the retractive forces of the lung increase. These effects of ventilation are fully reversed when the lungs are kept at constant inflation with air for 3 hr. In the present study (table 4) freshly excised lobes ventilated with air (VT = 30 ‘A TLV; f = 12/min; EEP = 0 cm H,O) for 3 hr at room temperature,
113
DISTENSION AND SURFACTANT RELEASE
did not r~ver~omplete~y when sub~quently kept at constant inflation of 30 cm H,O pressure with pure 0, for 3 hr. TABLE 4 Recovery from the effects of ventilation after static inflation with oxygen .-. -
P,p (cm I-W)
Control (5)
Vent. (air) (5)*
Vent. (air)+CI
30 20 15 10 5 0
98.2 +0.22 93.6kO.65 85.3k2.18 68.6 +4.06 44.4+ 3.54 8.2kO.32 __--.
96.5 kO.66’ 88.3fl.91’ 78.0+3.01+ 52.4i4.90’ 30.453.22’ 6.1 f0.91”
97.81tO.31’ 91.4+ 1.12” 81.9,2.35+ 61.2k3.86’ 39.5 +2.45++ 8.7+0.33 --
(0,) (S)**
--..-
Values are means + SE and indicate lobe air volume at different deflating pressures expressed as percent lobe air volume at 40 cm H,O transpulmonaty pressure. The deflation pressure-volume curves were obtained in lobes when freshly excised (control), after ventilation with air, and linaliy after same lobes were kept at constant inflation (CI) with pure oxygen. The lobes were always degassed prior to PV measurements (see Methods). Numbers in parentheses indicate the number of lobes studied. * Ventilated with air (VT = 30‘A of TLV; f = lymin; EEP = 0 cm H,O; duration = 3 hr). ** Following ventilation (as indicated above), the lobes were kept at constant inflation (P,, 30 cm H,O) with 100 od 0, for 3 hr. The lobe was also surrounded with 0,. ’ Different from controls (P < 0.05 - < 0.01). ‘+ Different from control (P < 0.10). Comparison of differences was made by means of paired r-test.
Acknowledgements The author expresses his deep appreciation to Dr. J. Thliveris for the EM studies
and to Adolph Wellemin, Walfried Jansen and Kwok-Tung Ghan for their excellent technical assistanee.
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Bra&e, G. (1949). Studies on lipids in the nervous system : total phosphorus determination. Rcta Physiol. &and. 63: 39-40.
Evans, C. L., F. Y. Hsu and T. Kosaka (1934). Utilization of blood sugar and formation of lactic acid by the lungs. J. Physiol. (Land.) 82: 41-61. Faridy, E. E., S. Permutt and R. L. Riley (1966). Effect of ventilation on surface forces in excised dogs’ lungs. J. Appl. Physiol. 21: 1453-1462. Faridy, E. E. and A. Naimark (1971). Effect of distension on metabolism of excised dog lung. J. Appf. Physd. 31: 31-37.
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E. E. FARIDY
Faridy, E. E. (1973). Effect of hydration and dehydration
on elastic behavior of excised dogs’ lungs.
J. Appl. Physiol. 34: 597-605.
Felts, J. M. (1965). Carbohydrate and lipid metabolism of lung tissue in vitro. Med. Thoruc. 22: 89-99. Fiske, C. H. and Y. Subbarow (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66: 375400.
Folch, J., M. Lees and G. H. Sloane-Stanley (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. Greenfield, L. J. and G. 0. -Kimmell (1967). Application of pneumatic techniques to surface tension determinations. The surfactometer. J. Surg. Res. 7: 276-297. Gribetz, I., N. R. Frank and M. E. Avery (1959). Static volume-pressure relations of excised lungs of infants with hyaline membrane disease, newborn and stillborn infants. J. Clin. Inuesr. 38: 21682175. Heinemann, H. 0. (1961). Free fatty acid production by rabbit lung tissue in vitro. Am. J. Physiol. 201: 607610.
Levey, S. and R. Gast (1966). Isolated perfused rat lung preparation. J. Appl. Physiol. 21: 313-316. Luft, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: 409414.
Mangold, H. K. (1961). Thin-layer chromatography of lipids. J. Oil Chem. Sot. 38: 708-727. Parker, F. and N. F. Peterson (1965). Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. J. Lipid Res. 6: 455-460. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Ceil Biol. 17: 208212. Richardson, K. C., L. Jarrett and E. Finke (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35 : 3 13-323. Sabatini, D. D., K. Bensch and R. J. Barnett (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17: 19-58.
