The effects of surface forces on the lungs

Brit. 07. Dis. Chest (t968) 62, I69.

The Effects of Surface Forces o n the Lungs I. C. S. NORMAND 1 Department of Paediatrics, University College Hospital Medical School, London WC I THE lung is made up of some 300 million alveoli in contact with the external atmosphere through a system of branching tubes. The alveolar surfaces are acutely curved with an average radius of 75 microns and their total area is approximately 7° square metres. Throughout the lung a thin fluid film is interposed between the ventilating gas and the epithelial surfaces so that there is a continuous and elaborately convoluted interface between gas and liquid phases within the lung. It is the purpose of this article to consider some of the physical forces which act on a structure arranged in this way and to describe how they may affect the mechanics of breathing. An outline will also be given of those unique properties of the lung whereby the mechanical problems imposed by surface forces acting over large and acutely curved surfaces are minimized. The subject has been extensively reviewed in recent years, for instance by Mead (I96I), Radford (I964) , Pattie (i965) , Clements and Tierney (I965) arid Avery and Said (I965) , and readers are referred to those articles for a detailed and comprehensive account of our present understanding of the subject. Surface Tension

Cohesive forces exist between the molecules of any liquid (Fig. I). Within the bulk of a liquid these forces are randomly and evenly distributed about each molecule and they have no net resultant force. However, they are asymmetrically distributed about the molecules at the surface of the liquid,

FIG. I. Intermolecular forces in the interior and at the surface of a liquid. At the surface these have a resultant force perpendicular to the plane of the surface 1In receipt of a grant from The Association for the Aid of Crippled Children, New York VOL. LXU4 (Receivedfor publication May z968) I

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producing a net resultant force acting in a direction perpendicular to the plane of the surface (Fig. I). As a result, the surface molecules tend to be drawn in towards the bulk of the liquid and so reduce the area of the surface to a minimum. The surface layer, therefore, behaves much as if it were a membrane under tension, and the force at a gas-liquid interface can be considered as a surface tension. It is customarily measured in units of dynes per era, pure water or o.9°~o NaC1 solution having a surface tension of 72 dynes/cm, and plasma a tension of approximately 55 dynes/cm. If a clean wettable object such as a strip of frosted platinum is dipped into water a meniscus rises up each face of the strip thereby fractionally increasing the surface area of the water. The effect of the surface forces in attempting to re-establish a minimum surface area will be to pull the strip vertically downwards with a force of 72 dynes along each I cm length of contact (Fig. 2). Thus with a strip 2 cm long, contact with the

m

FIG. 2. Surface forces acting on a wettable strip suspended vertically in water, represented here in cross-section, will pull the strip into the water with a force proportional to the surface tension and to the length of the strip in contact with the surface

surface will be made along both faces of the strip, a total length of 4 cm. I f the strip is suspended from some form of weighing device its apparent weight on 4x72 immersion into the surface will increase by - 7 g = o'29 g (I g is equivalent to 981 dynes under normal gravitational conditions). As will be seen later this principle is utilized in the modified Wilhelmy surface balance which has been widely used in determining the surface tension of lung extracts. The effect of surface forces when the interface is spherical, for example when a bubble of air is suspended in liquid, must also be considered. Because surface tension tends to reduce the area of an interface to a minimum there will be a progressive reduction in the area of the spherical interface, and hence in the volume of the bubble unless an opposing force is generated. Such a force could be provided by the increasing pressure within the bubble produced by the squeezing effect of the contracting surface on the contained gas. There is thus a relationship under equilibrium conditions between the surface tension

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and the pressure within the bubble, which is expressed by the equation derived by La Place: 2T Pr (Where P is the pressure difference across the interface, T is surface tension, and r is the radius of curvature of the surface.) The equation can be simply understood by reference to the accompanying diagram (Fig. 3) adapted from Mead (I96o). If we imagine that the bubble can be sliced in half, two forces will be acting on the segments.

FIG. 3" A spherical bubble suspended in water has hypothetically been sliced in half. Surface tension, exerted around the circumference, is balanced by the pressure within the bubble acting over the area of the slice

(I) Around the circumference, surface tension (T dynes/cm) will tend to bind the two halves together and will have a total magnitude of 27rrT dynes. (2) The pressure within the bubble (P dynes/cm2) will be acting over the area of the slice, tending to push the two halves apart with a total force of 7rr2P. Under equilibrium conditions their two forces must be equal, i.e. 2T 2rrrT = ~rr2P, whence P r

In more general terms this equation states that for flexible curved surfaces under tension, equilibrium is obtained only when the pressure within the 2T curved surface is greater than that outside by an amount equal to 7 "

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NORMAND M e c h a n i c a l E f f e c t s o f S u r f a c e T e n s i o n on t h e L u n g

Transpulmonarypressures Von Neergard (1929) measured the static pressure-volume (P-V) relationships of excised lungs which he inflated slowly with air and then gradually deflated. Subsequently, after all gas had been removed from the lungs in a vacuum jar, the process was repeated using a 7~o gum arabic solution to fill the lungs. He obtained results similar to those shown in Fig. 4 where it can be seen that lower pressures are required to fill lungs with liquid than with air. During liquid filling, interracial forces are eliminated and the shape of the pressure volume curve is under those circumstances determined only by the resistance to stretching of the elastic tissues. However, as yon Neergard concluded, the difference between the air and liquid P - V curves is a measure of the interracial forces in the lung.

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FIO. 4" Air ( .) and liquid (. . . . . . . ) pressure-volume curves of excised lungs. V4o = the volume of air contained by the lung at a transpulmonary pressure of 4° cm water. It approximates to the maximum volume of the lung. A, deflation curve of normal lung; B, deflation curve of unstable lung with abnormal surface tension characteristics. For comparison, curve c represents the pressure-volume relationships of an inflation and deflation with a tidal volume of air commencing at a lung volume corresponding to the functional residual capacity By using the La Place equation, it is possible to calculate the pressures required to maintain alveolar inflation in the presence of surface forces. I f it is assumed that the lung lining has a surface tension approaching that of plasma (55 dynes/cm), alveoli 75 microns in radius will collapse when the trans2x55 pulmonary pressure falls below 752xx 5I o5 - 4 dynes/cm2 o r 75 x I o - ~ x 98 I = 15 cm H 2 0 . For alveoli of smaller radius the pressures required to prevent collapse would be proportionately greater. However, transpulmonary pressures at the end of expiration are normally much lower than i5 cm H 2 0 . Von Neergard

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had suggested that alveolar surface tension may be lowered by the presence of surface active agents, but it was not until 1955 that the presence of an alveolar lining layer with a very low surface tension was demonstrated by Pattle. Although the exceptional stability of the foam of the frothy sputum of patients with pulmonary oedema must have been observed on many occasions, Pattie was the first to realize that this stability could only be due to the presence of a very low surface tension at the air-liquid interfaces of the foam. He then showed that bubbles squeezed out of small lung fragments into aerated water, and presumed to be carrying some of the alveolar lining film with them, diminished in size very slowly. From the rate at which their volume decreased he calculated that the surface tension of the film surrounding the bubbles was less than 0.06 dynes/cm (Pattle 1958 ). If this value represented the interracial tension at the alveolar surface, the pressures required to maintain patency of alveoli would be quite insignificant, amounting to 0.02 cm H 2 0 for alveoli of 75 microns radius. However Fig. 4 shows that approaching maximum lung volumes the difference betweenthe saline P - V curves and the meeting point of the three air P - V curves is approximately 25 cm H 2 0 and only fails to insignificant levels when the lung is deflated to about 5O~o of its maximum volume (deflation curve A). The discrepancy between Pattle's observations on the very low surface tension surrounding bubbles squeezed from the lung and the comparatively large forces apparently exerted by the surface when the lung is fully inflated was to a large extent explained by Clements (1957). He showed that if the surface tension of saline extracts of lung was continuously recorded in a modified Wilhelmy balance which permitted cyclic compression and expansion of the surface film (Fig. 9) the surface tension fell to levels below 5 dynes/cm on compression but rose to 4o-5 ° dynes/cm when the film was expanded (Fig. Io(a)). Assuming that the alveolar lining film behaves in a similar manner, during full expansion of the lung, alveolar surface tension will be high, whereas during deflation it may fall to very low levels. An alveolar lining film with these properties would account for the differences between the air and liquid pressurevolume curves of Fig. 4.

Alveolar stability Clements had been concerned with the problem of alveolar stability during lung deflation. He pointed out that if alveoli of different radius but having the same interfacial tension were in communication (Fig. 5) the La Place equation indicated that higher pressures would be exerted across the walls of the smaller alveoli which would therefore collapse while the large alveoli remained patent. Gruenwald (1947) had previously concluded that such a mechanism might be responsible for the pattern of atelectasis seen in the lungs of many infants dying in the newborn period in which air is present in a few distended airspaces while the rest of the lung is totally collapsed. A lung with a constant surface tension throughout would undergo progressive alveolar closure during deflation, with the smallest alveoli collapsing first; and it would have a pressure-volume curve similar to curve B in Fig. 4. There are instances in which alveolar closure

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Fro. 5. Two groups of alveoli of unequal size are in communication through their respective airways. If the surface tension of the lining layer surrounding both groups is the same, the pressure difference across the walls of the small alveoli will be higher than in the case of the larger alveoli, since P = 2T/r. The small alveoli would therefore collapse during deflation can be observed. Radford (196o) was able to produce this situation experimentally by instilling into the trachea of excised rat lungs a solution of the detergent Tween 20. This procedure has the effect of replacing the normal lung lining layer by a film of the detergent which has a constant surface tension of 20 dynes/cm at all degrees of surface film compression. The lungs treated in this way behaved as predicted with progressive closure of alveoli during deflation from maximal inflation. Although at high lung volumes tissue forces are important in limiting further lung expansion and in maintaining alveolar stability (Mead 1961) it is necessary to emphasize the essential contribution of the lung lining layer to the maintenance of alveolar opening during deflation to low lung volumes and at low transpulmonary pressures (Clements et al. 1961). This is achieved not only because of the very low surface tension of the alveolar film when it is compressed, but also because the variation of surface tension proportionally with different degrees of film compression allows airspaces of differing radii of curvature to be in communication through branching airways (Fig. 5).

Openingpressures Fig. 4 (inflation curve) shows that when a lung from which all air has previously been completely removed is inflated with air a pressure of approximately 15 cm H 2 0 is required before any air enters the lungs. Thereafter the lung rapidly inflates with only small increments of pressure until the limiting volume is reached. There is usually little further increase in volume if the inflating pressure is raised above 4o cm H 2 0 . Foetal lungs when first inflated with air behave in the same way. Close observation of the surface of the lung

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during first inflation from an airless state shows scattered terminal units ' popping' open in an apparently random manner and reaching peak volume almost as soon as they open, while neighbouring units remain collapsed. Fig. 6

L

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A

B

c

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FIO. 6. A model representing an airway and terminal airspace of a fluid-filled foetal lung during the first breath. T h e smallest radius of curvature of the interface is seen in c, at which point m a x i m u m inflating pressures will be required to overcome the surface forces. Thereafter the radius of curvature increases (9) and lung expansion will continue without any further increase in pressure until limited by the resistance of the elastic tissues

offers an explanation for this behaviour. It represents an airway and terminal airspace of a fluid-filled foetal lung during the first inflation with air and shows that the smallest radius of curvature of the interface occurs at the point where a hemispherical bubble is formed at the end of the terminal airway (Fig. 6 c). While air is moving peripherally down the airways the total area of the interface is rapidly expanding and the surface tension is likely to be high. The combination of minimum radius and high surface tension requires a large inflating pressure to expand the lung beyond the stage shown in Fig. 6 c. But once air enters the airspaces the radius again increases (Fig. 6 n) and the volume, for the same pressure, will rapidly increase until limited by rising tension of the surface and by the elastic recoil of the surrounding tissues. Opening pressures therefore are largely determined by the radius of the terminal airways and by the high tension of an expanded surface film. Consequently the shape of the inflation limb of the P - V curve does not differ greatly between normal lungs and lungs which have grossly abnormal surface properties due to a high minimum surface tension. Only when lungs have first been treated with detergent and have a constant surface tension of 20 dynes/cm, which is considerably less than the tension of the normal expanded alveolar film, are opening pressures likely to be lowered significantly (Gruenwald 1947). Alveoli that have been once inflated normally do not close at end-expiration, but retain a significant residual volume of gas, so that subsequent inflations only require small increases of pressure. However if there is marked alveolar instability with extensive alveolar closure during each deflation, as for instance in the lungs of infants with hyaline membrane disease, alveoli that have collapsed can only be reopened by the development of opening pressures

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at each breath; and the work of breathing is thereby greatly increased (Karlberg et al. I954).

Pulmonary transudation and lung liquid clearance In his original communication Pattle (I955) had suggested that if alveolar surface tension was high 'enough suction would be exerted to fill the alveoli with a transudate from the capillaries'. It seems more likely that when the surface film is closely applied to the alveolar epithelium, as is normally the case, the alveoli would first collapse and so obliterate the alveolar surface. An analogous situation, however, may arise during the first breaths of the newborn when air enters a lung already containing over I oo ml of lung liquid. In these circumstances the air-liquid interface may be separated from the alveolar epithelium by a fairly wide rim of liquid much as in Fig. 6 D, and fluid displaced from alveoli into the interstitial space of the lung by the transpulmonary pressures exerted during inflation could well be sucked back into the alveolus during expiration if the interfacial tension is sufficiently high. Humphreys et al. (I967) have suggested that this mechanism may be responsible for the delayed clearance of lung liquid from the lungs of immature lambs after the onset of ventilation, since these animals all had high lung-extract surface tensions.

Surface-active Agents: Pulmonary Surfactant The distinctive surface properties of the lung are due to the presence of surface-active agents in the alveolar lining film. The nature of the surface forces has been demonstrated in Fig. i. If the liquid contains in solution molecules between which the intermolecular forces are weaker than those of the solvent, the solute molecules will be attracted less strongly towards the interior of the liquid and will steadily accumulate at the surface. Moreover, because of their weaker cohesive forces, the tension of the surface layer that they form will be lower than that of the pure solvent. Molecules with such properties are said to be surface-active. Soaps, which are esters of long chain fatty acids, are typical surface-active compounds. When orientated at the surface of a soap solution the non-polar hydrocarbon end of the chain is situated in the air while the polar ester grouping, which is strongly hydrophilic, is immersed within the solution. The surface of the solution thus consists of a single layer of soap molecules arranged in a regular fashion and orientated perpendicularly to the surface much as the pile of a carpet (Fig. 7). However soaps are rather soluble and if the surface film is compressed or expanded soap molecules move rapidly out of or into the surface, with the result that there is no change in their density in the surface and hence in the surface tension of the solution. Surface-active agents can readily be demonstrated in extracts of mammalian lungs and in alveolar washings, but the unique properties of the lung surface appear to be due to the presence in the lining layer of large amounts of dipalmitoyl lecithin (DPL) (Klaus, Clements & Havel I96I). Lecithins consist of two long fatty acid chains linked to a strongly polar phosphoryl choline unit in a structure similar to that of the soaps considered above, but

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m

FIO. 7. Arrangement of surface-active molecules at the surface of a solution. The hydrophilic polar groups are represented by the open circles and the hydrocarbon chains by the vertical lines

far less soluble than they are. D P L is unusual amongst biological lecithins in having both chains consisting of fully saturated fatty acids. Clements (1967) has emphasized the special characteristics of the pulmonary surfaceactive agent or surfactant, in particular the stability of the surface monolayer at high film compression when the molecular density becomes so great that film collapse and disorganization might be expected; indeed he describes the lung surface film as having a quasi-crystalline structure. Abrams (1966) has shown that D P L occurs in the lung in a lipoprotein complex and Clements (1967) suggests that the presence of adjacent saturated fatty acid chains in the same lecithin molecule, together with a subfilm of protein, may be the main factors conferring stability on the surface layer at high film concentrations. Pure D P L itself and the extracted lipoprotein complex when layered onto surface balances which allow cyclic changes of film surface area yield loops very Similar to those obtained from extracts of normal lungs. The high proportion of saturated fatty acid chains and the unusually high concentration of palmitic acid within the mammalian lung emphasize the special significance of these compounds within the lung.

The Synthesis of P u l m o n a r y Surfactant A clearer picture of the epithelial lining of the lung has emerged since the application of electron microscopy. It consists predominantly of flattened cytoplasmic extensions of the Type I cells or membranous pneumonocytes. Scattered amongst these are larger, granular and more globular alveolar cells or granular pneumonocytes (Type II cell) (Fig. 8). These latter cells contain numerous osmiophilic granules which on electron microscopy have a characteristic lamellar structure, and are thought to represent structures associated with the storage and release onto the alveolar surface of pulmonary surfactant. The evidence for this rests mainly on the close association between the time of appearance of significant numbers of these structures in the developing mammalian foetus and the time that such lungs show active surface properties (Buckingham & Avery 1962 ). This close temporal relationship has been observed, with only occasional exceptions (Brumley et al. 1967) in all mammalian species studied.

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FIG. 8. Diagram of the epithelial lining of the lung of a newborn rat. The Type I cells (CA I) with their flattened cytoplasmic extensions and the globular Type II cells (CA II) containing osmiophilic lamellar inclusions can be readily seen. L: alveolar lumen, C: capillary lumen, GR: red cell, Mes: mesenchyme (By courtesy of Dr M. Campiehe, from Avery & Said, 1965)

The lung itself is a metabolically active organ capable of rapid fatty acid synthesis and of rapid incorporation of fatty acid into phospholipids, a process which has been shown by autoradiographic techniques to take place in the large alveolar cell (Buckingham et al. 1966). Recent studies have also shown that the lung, both in foetal and later life, is capable of active de novo synthesis of lecithin (Gluck et al. I967b ). A measure of the activity of the lung in this respect is provided by the work of Tierney, Clements and Trahan (i 967) who have estimated that the half-life of the surface-active lecithin is only 14 hours.

The Measurement of Lung Surface Properties Pressure-volume curves

Pressure volume curves similar to those shown in Fig. 4 can be obtained by using simply assembled apparatus. The shape of the inflation limb, as discussed above, is to a large extent determined by the critical opening pressure of the alveolar units and by the limiting alveolar volume. It therefore yields little information about the internal surface of the lung. The shape of the deflation limb after maximal inflation, however, provides a measure of the ability of lung units to resist collapse, and is a particularly sensitive index of alveolar stability at low transpulmonary pressures. In order to lessen the risk of rupture of airspaces by very high inflating pressures a limiting pressure of 35-4 ° cm H 2 0 may be adopted as shown in Fig. 4-

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Normal lungs retain at least 6O~o of the m a x i m u m volume at transpulmonary pressures as low as 5 cm H 2 0 and even at zero pressure 25~o of the maximum volume may be retained (Fig. 4 A). In contrast, lungs in which the surface-tension is abnormally high show airspace closure commencing at high pressures and increasing progressively, so that the volume retained at each pressure is significantly lower than for normal lungs (Fig. 4 B). In cases of hyaline membrane disease it is not unusual for lungs to be almost totally atelectatic at zero pressure.

Stability of bubbles Pattie (I958) has described in detail the behaviour of bubbles squeezed out of small fragments of normal lung into a drop of aerated saline hanging from a cover slip, and in particular has emphasized the extremely slow rate at which their volume shrinks. From this he has estimated that the surface tension surrounding these bubbles must be less than o. i dynes/cm. For quantitative assessment he has used a 'stability ratio' which is defined as the ratio of the surface area of a bubble at the end of a 2o min observation period to the surface area at the beginning of this period, a figure that can be obtained from the ratio of the squares of the diameters at these two times. A stability ratio above o.6 for bubbles whose initial diameter is in the range of 35-6o microns is considered normal. Pattle also observed that if bubbles from normal lungs were extruded into a hanging drop of water previously de-aerated by boiling, they exhibited the phenomenon of 'clicking'. This term is given to the cyclical changes of diameter such bubbles undergo when viewed microscopically and consists of a slow increase in diameter followed by a sudden contraction, or 'click', to progressively smaller diameters until the bubble finally dissolves and disappears. The reason for this phenomenon is that as the air in the bubble dissolves in the surrounding de-aerated water the bubble becomes progressively flatter and at the same time larger in diameter when seen from above. The bubble then suddenly resumes a spherical shape of smaller radius as the surface readjusts to the diminishing volume. This phenomenon is only seen in bubbles extruded from lungs with an active lining layer.

Surface tension measurements of lung extracts The theoretical considerations relating to alveolar stability during deflation that led Clements (I957) to measure the surface tension of saline extracts of lung while the area of the surface film was alternately compressed and expanded have already been considered. The type of Wilhelmy balance which he used is shown diagrammatically in Fig. 9. The lung extract is obtained by mincing 3-5 g lung with scissors in 5 ° m l of saline and the resulting mixture is then strained through gauze into a PTF~. trough. A mechanically driven paddle is arranged to move slowly back and forward in the trough so that the surface in front of the paddle, which is totally surrounded by a PTF~. ribbon, is reduced to 2 o ~ of the maximum area and then re-expanded, each cycle lasting Io to 15 min. The movement of the paddle, corresponding to a change in surface

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Fio. 9. Modified Wilhelmy surface balance for measuring the surface tension of lung extracts. The surface film to the right of the paddle is entirely surrounded by a flexible Teflon ribbon, not shown in the diagram. A motor slowly drives the paddle backwards and forwards in the trough, and this movement, corresponding to a change in area of the surface, is displayed on the y-axis of a recorder. Surface tension is measured by the pull on a platinum strip dipping into the surface, and is displayed on the x-axis of the recorder

area of the film, is recorded on one axis of a suitable recording device and the surface tension on the other axis. The type of plot obtained from normal and abnormal lungs after cycling for two to three hours is shown respectively in Fig. Io(a) and (b). The minimum surface tension on compression of surface films of extracts of normal lungs often falls below 5 dynes/cm and always below 15 dynes/cm. As soon as the area of the film begins to be expanded from full compression the orderly arrangement of the densely packed surface-active m o l e c u l e s is d i s r u p t e d a n d t h e s u r f a c e t e n s i o n rises v e r y r a p i d l y . T h e c o m pression and expansion limbs of the curves are widely separated and the loops d e m o n s t r a t e h y s t e r e s i s . T h e s a m e p h e n o m e n o n is a l s o r e s p o n s i b l e for p a r t o f the difference between the inflation and deflation limbs of the normal pressureI00

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Ftc. I o. Plots from an x - y recorder of surface area and tension of extracts of (a) normal lung, and (b) lung from an infant dying of hyaline membrane disease. Area is expressed as a percentage of the maximum area of the surface film

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volume curve shown in Fig. 4. Minimum surface tensions from lungs with abnormal surface properties are higher than 15 dynes/cm and usually above 2o dynes/cm. The compression and expansion limbs of these curves from lungs with abnormal surface properties are not widely separated and show little hysteresis. The simple mincing process used to obtain lung extracts is not a particularly efficient method of removing surface active material from the lungs, and Levine and Johnson (i 964) have drawn attention to the lower surface tensions obtained when mincing is carried out on inflated lungs than when extracts are obtained from portions of the same lungs but after they have been made atelectatic by degassing. It is sometimes found, particularly when lungs of immature stillborn infants are examined, that minimum surface tensions may not be obtained until cycling is continued for up to 24 hours (Levine & Johnson, 1964; Reynolds et al. 1965b). In these circumstances it is reasonable to assume that relatively small quantities of surface-active material are present in the extract and that only by prolonged aging and cycling will there be sufficient recruitment of molecules to the surface to produce low surface tensions. A general relationship undoubtedly exists between the amount of surface active material in the extract and the minimum surface tension, but no attempt has been made to define the precise quantitative relationship between the two.

Comparison of methods of measuring surface tension in the lungs All these methods can be used to categorize lungs broadly into those with normal and those with abnormal surface properties, and in these qualitative terms give reproducible results. Moreover when more than one method is used to examine the lungs the results between the methods are in close agreement (Howatt et al. I965; Humphreys & Strang i967; Gandy et al. i968 ). Much of the experimental work comparing methods has been carried out on the lungs of foetuses and newborns. The concentration of surface active material in the lung is higher in the mature foetus and newborn than at any other time of life, while in the immature foetus and in the infant with hyaline membrane disease the concentrations can be very low. It is not surprising therefore that clear-cut qualitative differences have been reported between normal and abnormal lungs whatever method of measuring surface propecties has been used. However in a number of experimental and clinical situations, as for example oxygen toxicity, pneumonia, or vagotomy, discrepant results and apparent species differences have been reported. This may be because present methods cannot detect small differences in lung surface properties. It might be thought that measurement by chemical methods of the concentration of known surface-active substances in the lungs would provide a more sensitive quantitative comparison, and indeed the relationship between surface tension measurements and total lung phospholipid (Adams et al. I965) , disaturated lecithin (Brumley et al. i967) and acetone precipitated lecithin (Gluck, Sribney & Kulovitch I967) has been clearly established. However these methods may fail to distinguish pulmonary surfactant at the alveolar

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surface, which is the only site where it is functionally important, from that elsewhere in the lungs. Functionally, the pressure-volume relationships of excised lungs are likely to correspond most closely with the action of the alveolar surface forces in life, and unless significant departures from normal can be demonstrated in these, abnormalities in surface properties detected by other methods should be interpreted with caution. However as an exceptionally simple screening technique, if other methods are not available for assessing lung surface properties, Gandy et al. (I968) have recently suggested the use of Patfle's 'clicking' method, which only requires the observation under a microscope of bubbles squeezed from fragments of lung into a drop of boiled water or saline hanging from the under surface of a cover slip.

Conditions Associated with Abnormal P u l m o n a r y Surface Properties Immaturity In all experimental animals studied detectable surface activity appears late in foetal life, for example at i8 days in the mouse (term 19 days), 29 days in the rabbit (term 31 days), and at I25-I3o days in the sheep (term I47 days). Moreover the appearance of pulmonary surfactant in the lung in significant quantities is associated with significant alterations in the metabolic pathways responsible for its synthesis (Gluck et al. ,967b). Animals delivered before these times are not viable, but if immature foetal lambs are delivered at I25 days they can be artificially ventilated and will survive for some hours. On histological examination their lungs show the typical appearance of hyaline membrane disease (Reynolds et al. I965a; Normand et al. I968 ). Information about the time of appearance of surface activity in normal human foetuses is for obvious reasons difficult to obtain, but studies of lungs from fresh still-births and from immature infants dying from non-pulmonary causes suggest that some surface activity can at times be detected as early as 24 weeks of gestation (Gruenwald I963; Reynolds et al. I965b ). Whether this finding is typical of all human foetuses is not known, but the almost invariable association between hyaline membrane disease and immaturity suggests that in man as in other species the quantity of surface-active material in the lungs of immature foetuses is likely to be low.

Hyaline membrane disease This disorder of premature babies is characterized by increasing respiratory difficulty from shortly after birth and by increasing cyanosis despite high concentrations of inspired oxygen. Physiologically these babies have large rightto-left shunts and very low lung compliance. The mortality approaches 4O~o and at necropsy the lungs are collapsed and liver-like. They show extensive atelectasis and such airspaces as remain patent are lined with eosinophilic hyaline membranes (Fig. 1 I). The lungs of infants dying during the height of the disorder invariably have a deficiency of pulmonary surfactant whether this is measured on a surface balance (Avery & Mead I959) , by pressure-volume

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curves (Gribetz, Frank & Avery 1959) , bubble stability (Pattle et al. 1962), absence of bubble clicking (Gandy et ah I968 ) or by chemical methods (Adams et al. 1965; Brumley, Hodson & Avery 1967). This deficiency can account for the clinical features. At end-expiration alveolar closure renders the lung largely atelectatic and opening pressures of 15-2o cm H 2 0 have to be applied at each breath to achieve any alveolar ventilation. The work of breathing is greatly increased and ventilatory insufficiency rapidly worsens. A high Pco2 and low Po2 in arterial blood cause a rise in pulmonary vascular resistance and hence the re-establishment of foetal pathways between the pulmonary and systemic circulations. Right-to-left shunts of over 75~o of cardiac output may develop (Strang & McLeish 196 i) with a disastrous breakdown of adequate alveolar gas exchange. Treatment at the present time is essentially supportive, with correction of the severe metabolic and acid-base disturbances that may occur together with the use of high inspired oxygen concentrations if the infants are hypoxaemic. Assisted respiration is being increasingly used in severely affected babies. Chu et al. (1967) have advocated the use ofacetyl choline as a means of reducing the pulmonary vascular resistance and improving pulmonary perfusion in severely hypoxic infants. These authors also noted some increase in pulmonary compliance after the administration of synthetic dipalmitoyl lecithin by aerosol, and in an earlier uncontrolled study Robillard et al. (1964) had obtained apparently encouraging results by this form of treatment. Such welcome attempts at a more fundamental approach to the treatment of this disorder are however still in their early stages and require further study before they can be adequately assessed.

Pulmonary artery ligation In experimental animals a loss of pulmonary surface activity in the appropriate lung lasting for several weeks follows within 24 hours' unilateral pulmonary artery ligation (Finley et al. 1964). Recovery takes place as the bronchial collateral circulation develops. Greenfield et al. (I967) have confirmed these findings and shown moreover that the changes are associated with abnormalities of both saline and air pressure-volume curves as well as with a reduction of disaturated lecithin concentration in the lungs.

Other conditions The above three conditions are the only instances unequivocally associated with a deficiency of pulmonary surfactant. In a variety of other experimental and clinical situations such as 02 and CO2 poisoning, cervical vagotomy, cardiopulmonary by-pass and pulmonary infections it has been suggested that there is impairment of normal pulmonary surface activity. There appear often to be considerable species differences, and in most cases pulmonary surface properties have not been examined by more than one method. It has been pointed out earlier that current techniques do not permit detection of minor variations in concentration of pulmonary surfactant and this may well account for the equivocal nature of many of these results.

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Focal and Post-operative Atelectasis If a lung extract is poured into a balance and its surface tension measured, values of 20 dynes/era or greater are recorded and the tension only falls to very low levels when the surface is compressed. Similarly when the surface is compressed and held in compression the tension gradually rises from very low levels towards the equilibrium value. It has been suggested (Clements 1962 ) that desorption from the surface occurs if it is left without alteration of its area for any length of time with gradual solution of the compressed surface molecules into the subjacent solvent. Re-expansion of the surface will recruit more surface-active molecules into the surface layer so that the tension will again fall to minimum values on recompression. Mead and Collier (i959) showed in anaesthetized dogs that either during spontaneous shallow breathing or when they were artificially ventilated with a small constant tidal volume, there was a progressive fall in pulmonary compliance with time which approached 5o7o of initial values after 2 hours. However these changes could be prevented by a single large breath every I O minutes. Somewhat similar results were obtained by Williams, Tierney and Parker (i966) when rabbit and newborn sheep lungs were ventilated at constant tidal volumes with low transpulmonary pressures (2 cm H20), but the fall in compliance was abolished when the transpulmonary pressure was raised to 5 cm H20, although the tidal volume was unchanged. In man, restriction of the chest wall by strapping reduces lung compliance even after the strapping has been removed, but compliance returns to normal after a deep breath has been taken (Caro, Butler & Dubois 196o). Similarly Ross et al. (I963) have noted a reduction in functional residual capacity following inguinal herniorrhaphy. The most likely explanation for these observations must rest on the theoretical considerations outlined above and suggests that areas of focal atelectasis can develop within normal lungs in which there is no general deficiency Of pulmonary surfactant. It does moreover suggest that the atelectasis seen in post-operative or unconscious patients is not due solely to the occlusion of airways by bronchial secretions, but may in part be due to alveolar closure taking place as a result of localized changes in pulmonary surface tension caused by shallow breathing accompanying post-operative pain or central respiratory depression. Certainly there are theoretical grounds for recommending regular deep breaths for such patients. It is also possible that part of the physiological role of spontaneous yawning and sighing is to reopen alveoli that may have closed during a previous period of quiet shallow breathing. Conclusion The configuration of the mammalian lung is such that potentially large surface forces can operate at the alveolar interface, and the effect of these is to cause closure of air-spaces during deflation and at low transpulmonary pressures. This situation is observed experimentally in lungs previously instilled

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FIG. I I. Section from the lung of an infant dying from hyaline m e m b r a n e disease. T h e r e is extensive atelectasis, a n d hyaline m e m b r a n e s line the few dilated airspaces

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with detergent and in the lungs of immature animals. Clinically it occurs in babies with hyaline membrane disease. However the potential effect of these surface forces is minimized in normal lungs by the presence of an alveolar lining layer with unique surface properties due to the presence of a lipoprotein containing a high proportion of dipalmitoyl lecithin. The surface tension of this layer falls on compression and rises when the area of the surface film is expanded. As a result great stability is conferred on the airspaces of the lung, and because of the very low tensions reached on compression alveoli do not collapse at transpulmonary pressures approaching zero. The alveolar epithelium is a metabolically active structure with specific pathways for the synthesis of surface-active compounds and mechanisms for their release onto the alveolar surface. So secure are these synthetic mechanisms in man that apart from the hyaline membrane disease of premature newborn infants, clinical syndromes clearly associated with significant deficiencies of pulmonary surfactant await description.

R~jCe~'ences ABRAMS, M. E. (1966) Isolation and quantitative estimation of pulmonary surface active lipoprotein. 07. appl. Physiol., 21, 718. ADAMS, F. H., FUJIWARA, T., EMMANOUILIDES,G. ~: SCUDDER, A. (I965) Surface properties and lipids from lungs of infants with hyaline membrane disease. 07. Pediat., 63, 537AVERY, i . E. & MEAD, J. (1959) Surface properties in relation to atelectasis and hyaline membrane disease. Amer. 07. Dis. Child., 97, 517 • AVERY, M. E. & SAID, S. I. (1965) Surface phenomena in lungs in health and disease. Medicine (Baltimore), 44, 503 • BRUMLEY, G. W., CHERNIK, V., HODSON, W. A., NORMAND, C., FENNER, A. & AVERY, M. E. (1967) Correlations of mechanical stability, morphology, pulmonary surfactant and phospholipid content in the developing lamb lung. 07. din. Invest., 46, 863. BRUMLEY, G. W., HODSON, W. A. & AVERY, M. E. (I967) Lung phospholipids and surface tension correlations in infants with and without hyaline membrane disease and in adults. Pediatrics, 40, I3. BUCKI~OHAM, S. & AVERY, M. E. (1962) Time of appearance of lung surfactant in the foetal mouse. Nature (Lond.), i93 , 688. BUCKINOHAM, S., HmNEMANN, H. O., SOMM~RS, S. C. & MCNARY, W. F. (1966) Phospholipid synthesis in the large pulmonary alveolar cell. Amer. 07. Path., 48, IO27. CARO, C. G., BUTLER, J. & D u BOlS, A. B. (I96O) Some effects of restriction of chest cage expansion on pulmonary function in man: an experimental study. 07. din. Invest., 39, 573. C s u , J., CLEMENTS,J. A., COTTON, E. K., KLAUS, M. H., SWEET, A. Y. & TOOLEY, W. H. (I967) Neonatal pulmonary ischaemia. Part I: Clinical and physiological studies. Pediatrics, 4o, 7o9 • CLEM~NTS, J. A. (i957) Surface tension of lung extracts. Proc. Soc. exp. Biol. (N.T.), 95, I7O. CLEMENTS, J. A. (1962) Surface phenomena in relation to pulmonary function. Sixth Bowditch Lecture. Physiologist, 5, I I. CLEMm~TS, J. A. (1967) In The Development of the Lung. Eds. A. V. S. de lZueck & R. Porter. p. 202. London: Churchill. CLEMENTS,J. A., HUSTEAD, R. F., JOHNSON, R. P. & GRIEETZ, I. (1961) Pulmonary surface tension and alveolar stability. 07. appl. Physiol., i6, 444. CLEMENTS, J. A. & TIERNEY, D. F. (1965) In Handbook of Physiolog2. Respiration II. Eds. W. O. Fenn & H. Rahn. p. 1565. Washington: American Physiological Society. V O L . LXII

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FINLEY, T. N., TOOLEY, W. H., SWENSON, E. W., GARDNER, R. E. & CLEMENTS,J. A. (I964) Pulmonary surface tension in experimental atelectasis. Amer. Rev. resp. Dis., 89, 372. GANDY, G., BRADBROOKE,J. G., NAIDOO, B. T. & GAIRDNER, D. (I968) Comparison of methods for evaluating surface properties of lung in perinatal period. Arch. Dis. Childh., 43, 8. GLUCK, L., MOTOYAMA,E. K., SMITS, H. L. & KULOVICH, M. V. (I967a) The biochemical development of surface activity in mammalian lung. I. The surface active phospholipids; the separation and distribution of surface-active lecithin in the lung of the developing rabbit fetus. Pediat. Res., x, 237. GLUER, L., SRIBNEY, M. & KULOVICH, M. V. (i967b) The biochemical development of surface activity in mammalian lung. II. The biosynthesis of phospholipids in the lung of the developing rabbit fetus and newborn. Pediat. Res., x, 247. GREENFIELD, L.J., CHERNICK, V., HODSON, W. A. & BRUMLEY, G. W. (I967) Alterations in pulmonary surfaetant following compression atelectasis, pulmonary artery ligation, and reimplantation of the lung. Ann. Surg., x66, Io 9. GRIBETZ, I., FRANK, N. R. & AVERY, M. E. (I959) Static volume-pressure relations of excised lungs of infants with hyaline membrane disease, newborn and stillborn infants. 07. din. Invest., 38, oi68. GRUENWALD, P. (I947) Surface tension as a factor in the resistance of neonatal lungs to aeration. Amer. 07. Obstet. Gynee., 53, 996. GRUENWALD, P. (I963) Normal and abnormal expansion of the lungs of newborn infants obtained at autopsy. II. Opening pressure, maximal volume and stability of expansion. Lab. Invest., x2, 563 . HOWATT, W. F., AVERY, M. E., HUMPHREYS, P. W., NORMAND, I. C. S., REID, L. & STRANG, L. B. (I965) Factors affecting pulmonary surface properties in the foetal lamb. Clin. Sci., 29, 239. HUMPHREY', P. W., NORMAND, I. C. S., REYNOLDS, E. O. R. & STRANG, L. B. (I967) Pulmonary lymph flow and the uptake of liquid from the lungs of the lamb at the start of breathing. 07. Physiol. (Lond.), 193, I. HUMPHREYS, P. W. & STRANG, L. B. (I967) Effects of gestation and prenatal asphyxia on pulmonary surface properties of the foetal rabbit. 07. Physiol. (Lond.), x92, 53. KARLBERO, P., COOK, C. D., O'BRIEN, D., CHERRY, R. B. • SMITH, C. A. (I954) Studies of respiratory physiology in the newborn infant; observations during and after respiratory distress. Acta Paedlat. (Uppsala), 43, 397KLAUS, M. H., CLEMENTS,J. A. & HAVE~, R . J . (i96i) Composition of surface active material isolated from beef-lung. Proc. nat. Acad. Sci. (Wash.), 47, I858. LEVINE, B. E. & JOHNSON, R. P. (I964) Surface activity of saline extracts from inflated and degassed normal lung. 07. appl. Physiol., x9, 333. MEAD, J. (i96o) Elementary considerations concerning influence of surface tension on the mechanical behavior of the lungs and in particular the stability of the air spaces. Amer. Rev. resp. Dis., 8I, 739MEAD, J. (i96i) Mechanical properties of lungs. Physiol. Rev., 4x, 28I. MEAD,J. & COLLIER,C. (I959) Relation of volume history of lungs to respiratory mechanics in anesthetized dogs. 07. appl. Physiol., x4, 669. YON NEERGARD, K. (I929) Neue Auffassungen fiber einen Grund-begriff der Atemmechanik. Die Retrakfionskraft der Lunge, abh~ingig vonder Oberfl/ichenspannung in den Alveolen. Z. ges. exp. Med., 66, 373NORMAND, I. C. S., REYNOLDS, E. 0. R., STRANO, L. B. & WIGGLESWORTH,J. S. (I968) Flow and protein concentration of lymph from the lungs of lambs developing hyaline membrane disease. Arch. Dis. Childh., 43, 334. PATTLE, R. E. (I955) Properties, function and origin of the alveolar lining layer. Nature (Lond.), I75, I I25. PATTLE, R. E. (I958) Properties, function and origin of the alveolar lining layer. Proc. roy. Soy. B, I48, 217.

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PATTLE, R. F.. (I965) Surface lining of lung alveoli. Physiol. Rev., 45, 48. PATTLE, R. E., CLAIREAUX, A. E., DAVIES, P. A. & CAMERON, A. H. (I962) Inability to form a lung-lining film as a cause of the respiratory distress syndrome of the newborn. Lancet, ii, 469. RADFORD, E. P. (I96o) Mechanical factors determining alveolar configuration. Amer. Rev. resp. Dis., 8I, 743RADFORD, E. P. (i964) In Handbook of Physiology. Respiration L Eds. W. O. Fenn & H. Rahn. p. 429. Washington: American Physiological Society. I~YNOLDS, E. O. R., JACOBSON, H. N., MOTOYAMA,E. K., KIKKAWA, Y., CRAIG,J. M., ORZALESI, M. M. & CooK, C. D. (I965a) The effect of immaturity and prenatal asphyxia on the lungs and pulmonary function of newborn lambs: the experimental production of respiratory distress. Pediatrics, 35, 382. REYNOLDS, E. O. R., ORZALESI,M. M., MOTOYAMA,E. K., CRAIG, J. M. & COOK, C. D. (I965b) Surface properties of saline extracts from lungs of newborn infants. Acta Paediat. (Uppsala), 54, 51 I. ROEILLARD, E., ALARIE, Y., DAGENAIs-PERuSS~, P., BARIL, P. & GUXLBV.AULT,A. (I964) Micro-aerosol administration of synthetic fl-y-dipalmitoyl-L-a lecithin in the respiratory distress syndrome. A preliminary report. Canad. med. Ass. 3 , 9o, 55. Ross, J. C., ELLER, J. L., GLENN, D. L. & KING, R. D. (I963) Alterations in lung volume and intrapulmonary gas mixing after inguinal herniorrhaphy in patients with normal lung function and in patients with emphysema. Amer. Rev. resp. Dis., 88, 213. STRANO, L. B. & McL~IsH, M. H. (I96I) Ventilatory failure and right-to-left shunt in newborn infants with respiratory distress. Pediatrics, 28, 17. TIERNEY, D. F., CLEMENTS,J. A. & TRAHAN, H . J . (I967) Rates of replacement of lecithins and alveolar instability in rats. Amer. o7. Physiol., 213, 67 i. WILLIAMS, J. V., TIERNEY, D. F. & PARKER, H. R. (I966) Surface forces in the lung, atelectasis and transpulmonary pressure, ft. appl. Physiol., 2x, 8I 9.