This dissertation has been fi7—1 A microfilmed exactly as received ° BECKMAN, David Lee, 1939- SURFACTANT, PRESSURE-VOLUME CURVES, AND COMPONENTS OF LUNG COMPLIANCE IN RATS: EFFECTS OF EXPOSURE TO ONE ATMOSPHERE OF OXYGEN.

The Ohio State University, Ph.D., 1967 Physiology

University Microfilms, Inc., Ann Arbor, Michigan David Lee Beckman 1967

All Rights Reserved SURFACTANT, PRESSURE-VCLUME CURVES, AND COMPONENTS

OF LUNG COMPLIANCE IN RATS: EFFECTS OF EXPOSURE

TO CNF ATMOSPHERE OF OXYGEN

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

David Lee Seckrian, B.Sc., K. Sc

The Ohio State University 1967

Approved by

Advis ar Department of Fhysiology College of Medicine ACKNOWLEDGMENTS

I especially wish to express my sincere appreciation to my advisor, Dr. Harold S. Weiss, who provided guidance and leadership in this project through his advice, patience and interest. The example he has shown as a scientist will be remembered far beyond the completion of this study.

To Dr. Edwin P. Hiatt I owe a special debt of gratitude for the encouragement and enthusiasm which he has offered during my graduate training. He was responsible for my first interests in the area of environmental physiology and has continually given his support to this research project.

Sincere appreciation is extended to Dr. Ronald A. ’.-/right, Dr.

Rodney A. Rhoades and Capt. Richard Pilmer for their interest, suggest­ ions, and help in the laboratory.

In addition I would like to thank Charles R. Wharton, E. Sue

Xreglow and Joseph F. Pitt for their technical assistance.

ii VITA

Ray 11, 1939 Born - Dayton, Ohio

1962 ...... B.Sc., The Ohio State University, Columbus, Ohio

1962~1964 .... Teaching Assistant and Research Fellow, Ohio State University, Columbus, Ohio

1 9 6 4 ...... M.Sc., Department of Physiology, Ohio State University, Columbus, Ohio

1965-1967 .... National Aeronautics and Space Administration Trainee

PUBLICATIONS

Weiss, H.S., Beckman, D.L., Wright R.A. Delayed Mortality in Adult Chickens Exposed to 1 Atmosphere Oxygen. Nature, 128:1003, 1965.

Beckman, D.L. and Weiss, H.S. Pulmonary Surfactant and Fressure- volume Curves in 0^ Poisoned Rats. Federation Proc. 26-2, 196?.

Beckman, D.L. and Weiss, H.S. Effects of Oxygen Toxicity on Pressure- volume Curves, Lung Compliance and Surfactant. (In preparation)

Beckman, D.L. and Weiss, H.S. Effects of Small Amounts of Nitrogen on Rat Survival in High Po9. (In preparation)

Beckman, D.L. and Scarpelli, D.G. Effects of Diamox on the Ultra­ structure of Rat Kidney Tubules. (In preparation)

FIELDS CF STUDY

Major Field: Physiology

Studies in Renal Physiology. Professor Edwin P. Hiatt and Dante G. Scarpelli

Studies in Environmental Physiology. Professors Harold 3. Weiss and Edwin P. Hiatt CONTENTS

Page

ACKNOWLEDGMENTS...... ii

VITA ...... iii

FIGURES...... vi

T A 3 L E S ...... viii

INTRODUCTION ...... 1

HISTORICAL REVIEW

The Surface Lining of Lung Alveoli...... 3 Surface tension and lung mechanics...... 6 Methods for studying surfactant...... 9 The surface lining in several different species. . . 11 The lung lining in various pathological conditions . 1 2

Mechanical Properties of Lungs Related to Surface Tensions ...... 15 Mechanical behavior of the lungs under nearly static conditions ...... 15 Surface tension and pressure-volume curves...... 16 Role of tissue forces on pressure-volume curves . . 18 Dynamic volume-pressure curves and compliance . . . 18

The Pulmonary Manifestations of Oxygen Toxicity . . . 19 O^rgen at one atmosphere...... 19

METHODS

General Procedure ...... 22 A n i m a l s ...... 23 Oxygen E x p o s u r e ...... 23 Removal of Rat Lungs ...... 25 Pressure-volume Apparatus; Air I n f u s i o n...... 26 Saline I n f u s i o n ...... 31 Residual Air in L u n g s ...... 31 Perfusion of Lungs to Remove Surfactant...... 33 Pressure-volume Curves . 37

iv CONTENTS (Continued)

Page

Rate of Filling; Maximum Pressures; Volumes...... 37 Perfusate Surfactant Measurements...... 41 Lecithin Extraction Procedure...... 41 Thin Layer Chromatography - Separation of Lecithin . . . 43 Statistical Analysis of D a t a ...... 50

RESULTS...... 52

Oxygen Poisoned Rats ...... 62 Comparison of Normal and Oxygen Poisoned R a t s ...... 70

DISCUSSION

General ...... 34 Effects of Oxygen Toxicity on Pulmonary Surfactant . . . 85 Effects of Oxygen Toxicity on Tissue Compliance ..... 91 Lung Lecithin Values as an Index to Surface Activity. . . 91 Methods of Extraction of Surfactant ...... 93 Effect of Edema on Air and Saline Pressure- volume Curves...... 96 Oxygen Toxicity and Atelectasis - Lung Residual Volumes ...... 99 Static and Dynamic Compliance Measurements ...... 100 Method of Lipid A n a l y s i s ...... 100 Saline Inflation and Deflation Curves...... 101

SUMMARY...... 103

BIBLIOGRAPHY...... 105

v 1

FIGURES

Figure Page

1. Oxygen Chamber...... 24

2. Air-Filled L u n g ...... 27

3. P-V Apparatus...... 28

4. Block Diagram of Apparatus for Automatic Regis­ tration of Pressure Volume Curves from Excised Rat Lungs ...... 29

5. Saline Filled L u n g ...... 32

6. Perfusion Apparatus ...... 35

7. Block Diagram of Apparatus for Collection of Pulmonary Surfactant via Perfusion of Pulmonary Circulation ...... 3&

8. X-Y Recorder...... 39

9. Six Successive Air P-V Curves on an Excised Rat L u n g ...... 53

10. Air P-V Curve from a Control Rat Before and After Perfusion to Remove Surfactant - Curves 5 and 6 Shown...... 54

11. Summary - Control Pressure - Volume Curve Before and After Perfusion ...... 55

12. Complete Sequence of P-V Curves from a Control Rat . . 58

13. Control Air and Saline P-V C u r v e s ...... 60

14. Control Air and Saline P-V C u r v e s ...... 61

15. Air P-V Curve from an Oxygen Exposed R a t ...... 64

16. Summary - Oxygen Pressure - Volume Curve Before and After Perfusion...... 66

vi FIGURES (Continued)

Figure Page

17. Complete Sequence of P-V Curves - Oxygen Exposed R a t ...... 69

18. Before Perfusion Air Curves...... 71

19. After Perfusion Air C u r v e s ...... 72

20. Surface Compliance - Lecithin ...... 82

vii TABLES

Table Page

1. Statistical Analysis of Control P-V Curves...... 57

2. Control Saline pressure-Volume Curves ...... 63

3. Statistical Analysis of Oxygen P-V Curves ...... 67

k. Oxygen Saline Pressure-Volume Curves ...... 68

5. Statistical Analysis of Air Curves - Comparison of Control and Oxygen Exposed Rats, Complete Sequence ...... 73

6. Statistical Analysis of Air Curves - Comparison of Control and Oxygen Rats, All R a t s ...... 7^

7. Statistical Analysis - Control and Oxygen Comparison of Paired Differences, 12 R a t s ...... 75

8. Statistical Analysis of Saline Curves ...... 77

9. Surface Compliance Values ...... 78

10. Comparisons Between Control and Oxygen Exposed Rats of Body Weight, Lung Weight, Buoyancy, and Lecithin ...... 80

viii INTRODUCTION

Oxygen poisoning has been studied extensively (see reviews by

Bean, 1945; Roth, 1964) but no completely satisfactory explanation appears available for the observed effects on either lungs or other tissues. During exposure to one atmosphere of oxygen, respiratory distress is frequently seen, and on post mortem atelectasis, edema and alveolar thickening is frequently reported, suggesting that the lungs may be a primary target. Oxygen toxicity increases lung

"stiffness" (Bean, 1945), hut it has not been shown if this change is due to increased tissue rigidity, increased surface tension or both. Increases in surface tension via loss of surfactant would appear to be a likely mechanism for atelactasis and edema at least, but the data is controversial (Pattle, 1965; Fujiwara et al.. 1964;

Kennedy, 1966; Bondurant and Smith, 1962; Giammona, 1965; Collier et al.. 1965). Some evidence indicates that there are species differences. Giammona, Kemer and Bondurant (1965) for example, reported that oxygen toxicity resulted in high lung surface tension in cats and rabbits but not in rats. Fujiwara (1964 ) found no effect of oxygen on surfactant or surface tension in guinea pigs.

However, it has also been suggested that the method of surfactant extraction may account for the variance in results. Giammona et al.

(1965) and Levine and Johnson (1964) showed that the removal of surfactant by mincing or chopping the lungs used in many reported

1 2 experiments, produces considerably less surfactant than homogenization and lavage.

It was felt that further knowledge of the mechanisms by which high concentrations of oxygen produce pulmonary damage would be ob­ tained by applying more precise techniques than used before in the measurement of surfactant and surface tension changes in rat lungs.

The rat should serve as an ideal animal for such work because of its convenient size and susceptibility to oxygen toxicity. The techniques chosen to elucidate the nature and cause of the lung damage included!

1) use of air and saline pressure-volume curves to separate tissue

effects from effects solely due to surface tension, 2) extraction of

surfactant by perfusion rather than mincing, 3) quantitation of the

extracted surfactant by thin layer chromatography, and 4) correlation of the amount of surfactant extracted with compliance changes in the lungs. HISTORICAL REVIEW

The Surface Lining of Lung Alveoli

The fact that the lung has an insoluble lining film was in­ ferred, in the first instance, from properties of foam obtained from the lungs (Pattle, 1955)* In acute lung edema (Pattle, 1956), or when saline is poured into the trachea in vivo, or after excision of the lungs (Pattle, 1958), copious froth is obtained. The froth obtained by these means was found to have many peculiarities. One of the first of these was the resistance of the froth to chemical antifoams such as silicone antifoam and octyl alcohol (Pattle, 1955,

1958). These same antifoams, however, completely destroy the froth obtained from shaking edema fluid, blood or other body fluids. Re­ sistance to antifoams was closely linked to surface tension character­ istics (Pattle, 1958) and consequently further studies were started in the area.

The behavior of bubbles was studied by washing froth obtained from lungs with distilled water (Pattle, 19&5)• process can be repeated without altering either the resistance of the froth to anti­ foams or the low surface tension which is normally developed. Thus it can be concluded that the froth is insoluble in water. If a bubble is dissolved in air-free water, it leaves behind a trans­ parent "ghost". Thus the bubbles of the lung foam possess a lining film of solid character which is highly insoluble. Another character- istic peculiar only to foam bubbles from the lung is the phenomenon known as "clicking" (Pattle, 1965). Viewed on a microscope slide these bubbles expand slowly and then suddenly contract by about

10 percent of their diameter. This process continues with each contraction slightly later than the previous expansion. Bubbles from lung foam last much longer than bubbles obtained from shaking blood or edema fluid.

The chemical characteristics of the surface lining material of lungs has been studied extensively. Some of the original work in this area was done by Klaus, Clements, and Havel (1961) on the beef lung using the tracheal transudate from the perfusion of the pul­ monary artery. They found that when the dried powder was spread on water in a Langmuir trough that it lowered surface tension to less than 10 dynes per square cm. and that the powier contained 50 to

70 percent lipids and 5 percent nitrogen. The lipids were about

percent phospholipids. Using the lavage technique, bubbles from a cow lung were washed with water and analyzed by infra-red absorp­ tion spectroscopy (Pattle and Thomas, 1961). The spectrum was found to be qualitatively identical to that of purified egg lecithin.

It also suggested that the quantity of protein in the bubble lining was about 1 percent. Small amounts (less than 1$) of other lipids may have been present. 3rown (1962) reported that the surface active material from mammalian lungs was quantitatively precipitated with trichloroacetic acid. Choline was found which indicated that the 5 surface material was alpha lecithin. Recent studies have gone more thoroughly into the quantitation of lipid components of the surface layer. A major difficulty in the interpretation of the results of these studies has been the influence of the method of surfactant removal. Levine (196*0 compared minimum surface tensions of ex­ tracts obtained by chopping, mincing, and homogenation of both aerated and degassed lungs, and found that in each case aerated lungs showed low minimum surface tensions, but that degassed lungs showed low minimal surface tensions only in the case of homogeniza­ tion. Results using minced lung from rabbits (Fujiwara, 1964) showed that phospholipids constituted 61 percent of the total lipids and that the next highest group was that of triglycerides which amounted to 19 percent of the total lipids. It was reported that lecithin constituted 48 percent of the phospholipids. Using the lavage technique in dogs, Morgan et al. (1965) reported that the phospholipid fraction constituted 74 percent of the total lipids, and that lecithin amounted to 55 percent of the total lipids. A further analysis of whole lung lipids in dog lungs showed the phos­ pholipid fraction of the total lipids to be 70 percent, and the lecithin fraction to be 41 percent.

The saline-dispersible lining complex or alveolar surfactant is not the same thing as the insoluble surface film which lines the alveoli and forms the froth (Pattle, 1965). The production of this insoluble surface film which lines the alveoli is probably formed

from the saline-dispersible "lining complex". This complex is the 6 substance which is normally removed by lavage techniques. Apparently a natural lining film is formed in vitro from the substance removed by lavage techniques. Even the fluid squeezed from atelectic lungs demonstrated the normal surfactant characteristics of "clicking"

(Pattle, 1965). All evidence points to the presence in the alveoli of a saline-dispersible lipoprotein complex, rich in phospholipids and of high molecular weight, from which the insoluble lining film is formed by some kind of surface action.

Surface Tension and Lung Mechanics

The action of surface tension was described by Clements (19^2)

The molecules at the surface are attracted more strongly to their neighbors below the surface and are attracted only weakly to the sparser population of molecules in the air above the surface. Because the net pull is downward the surface molecules tend to dive and the surface shrinks to the least possible area.

By virtue of this tendency to contract a curved surface exerts a pressure towards its concave side.

Von Neergaard (1929) is credited with the original work on lung surface tension although he did not discover the substance responsible for lowering it. He found that the retractive force of a lung deflating when filled with air was greater than that of a lung filled with gum arable solution. He also measured surface tension by a technique which indicated the force required to pull a stirrup away from the fluid surface. This method differs somewhat from present techniques in that in this case the surface tension is measured only when the surface is a state of extension. Mid-range 7 values were obtained (35-^6 dynes/cm.). The extremes can be measured only with a technique such as the Wilhelmy balance which measures the downward pull on a stationary plate. Von Neergaard considered the

formation of a bubble at the end of a capillary tube as an analog

for the surface geometry within the lungs. Such a bubble wculd be a segment of a sphere, and hence would exert a pressure in dynes/cm. according to the relationship P = 2y/r^, where y = coefficient of

surface tension in dynes/cm. and r = effective radius (cm.). As the bubble was formed, r would decrease to a minimum value when the bubble radius equaled the radius of the opening, and then would

increase again with further expansion. The pressure wculd have a maximum corresponding to the minimum radius of the bubble. He

considered the alveoli as spherical segments which only at maximum

inflation approached hemispherical shape and never, except in path­ ological states, exceeded it (Mead, 1961). In normal range of pressure-volume curves, the hemispherical shape is not exceeded.

When it is exceeded, pressure and volume are inversely related; a

state of instability is reached. Neergaard (as reported by Mead,

1961) showed on sheep, pig, and human lungs by fluid filling that:

(1) surface forces as opposed to tissue forces accounted for from

2/3 to 3 /^ of the total retractive pressure of the lung; (2) in the volume range of normal expiration, tissue pressure fell to zero;

(3) surface retractive pressures (the tendency towards deflation)

increased as lung volume increased. Thus the pressure required to 8

inflate a lung is primarily due to surface tension and to a lesser

extent to tissue resistance.

To summarize Neergaard’s contributions, he showed that surface

forces account for a large part of the retractive force of excised lungs. He suggested the bubble on the end of a capillary as a model

for an alveolus at the end of an alveolar duct. He said that alveoli were, in all physiological states, less than hemispheres. In his view, as the lung expanded, the effective radius of the alveoli

decreased resulting in the increased surface retractive forces (Von

Neergaard, 1929).

More recently surface tension has been studied with a variety of related techniques. Clements measured the surface tension of lung

extracts with a Wilhelmy balance and a Langmuir-Adam trough, so that

the surface could be compressed while measurements were being taken

(Clements, 1956). He discovered that the surface tension of lung

extracts could decrease to values as low as 10 dynes/cm. Avery (1962)

pointed out that if surfactant had a minimum surface tension of

55 dynes/cm. as found for blood serum, the lung would require a very

high pressure to keep it open, and the surface tension would cause a transudation of blood or edema fluid into the alveoli. Clements

et al. (1961) made the first measurements on lung extracts, plotting

surface tension against area which was altered cyclically. The

surface tension was reported to vary from 5 dynes/cm. to ^0 dynes/cm.

and the graph depicted a well-marked "hysteresis loop". Other sub­

stances, however, such as detergents produced a constant surface 9 tension and the characteristic hysteresis loop was not present. They expressed the variation of surface tension with area as "surface elastance", s, where:

s = AdiT /dA and A = area of the unit (cm^), Y~ = coefficient of surface tension in dynes/cm. They also calculated that an alveolus will remain stable in terms of its radius (cm), surface tension ( in dynes/cm), and surface compressibility if the pressure exceeds a certain minimum

P-j (dynes/cm2) given:

P! = (8 r - ks) (3r) where s = surface elastance as defined previously. Stability is thus favored by a high internal pressure, large radius, low surface ten­ sion, and high surface elastance. Most recent findings on the characteristics of surface tension lend support to this model.

Methods for Studying Surfactant

1. Several different but related methods have been used to study the lung lining. The most direct method (Pattle, 1965) is the plotting of pressure-volume carves, requiring not only an air in­ flation, but also a liquid filled lung to properly separate out the surface forces from other conditions such as fibrosis, consolidation, bronchial obstruction and edema. Rosenberg et al. (1962) carried out the complete procedure on rats which had inhaled an aerosol of I

10 aluminum oxide and metal dust. The state of the pressure-volume curve has been correlated with the state of the lining film by Clements

(1961) and Gruenwald (1962).

2. The examination of bubbles squeezed from a fragment of a lung has been used extensively with success by Pattle (1965). The bubbles from a lung fragment are squeezed onto a microscope slide and examined. The change in diameter over a 20 minute period is observed.

The ratio of the original to the final surface area is calculated and the result expressed as the "stability ratio". Pattle and Burgess

(1961) reported the slightly surprising result that edema did not effect the stability ratio except by raising it which would indicate a stable surface film.

3. Methods involving extracts of the lung have bean used extensively recently. These methods contain the complex in disper­ sion or solution. The extract can be analyzed in terms of lipid components (Morgan et al.. 1965; Harlan et al.. 1966; Fujiwara et al..

1964), the stability ratio can be determined (Pattle, 1965). or the surface tension measured on a Wilhelmy balance (Clements et al..

1961; Gruenwald et al.. 1961).

4. The most precise method of removing the contaminate free surfaoe layer is by means of lavage (washing saline in and out of the lung several times) or by transudation of fluid across the alveoli.

The lavage technique has been used extensively. Its relative merits have been compared to other processes by Levine and Johnson (1964).

The transudation process was introduced by Bondurant (1962). This 11 technique which involves perfusion of the pulmonary artery and col­ lection of the effluent foam from the trachea removes more surfactant from the lung than the aforementioned techniques.

The Surface Lining in Several Different Species

A variety of conflicting reports on the presence or absence of a surface lining complex in various species have recently been published. It was initially found (Pattle, 1958) that bubbles from pigeon and frog lungs were as stable as those from mammalian lungs.

Nevertheless Miller and Bondurant (1961) concluded from measurements on saline extracts of amphibian, reptile, bird, and mammal lungs, that "the presence of distinctive surface-active material was limited to mammalian lungs." On re-examination Pattle and Hopkinson (1963) found the behavior of bubbles from chicken lungs to be identical to those of mammal lungs. They found the bubbles from reptile and amphibian lungs to be somewhat different. They suggested that reptile and amphibian lungs have as good a lining film as do mammal lungs, but that the reserve of the saline-dispersible complex from which the surface film is derived was smaller. This conclusion was drawn from the finding that bubbles from squeezed lung were stable, but those from the lung washings were not stable. Tooley and Clements

(1962) proposed that the surface lining layer came from mitochondrial lamellar forms in the alveolar epithelial cells. They found these forms present in the lungs of the rat, cat, mouse, rabbit, dog, and man, but not in toad and pigeon lungs. The presence of lamellar 12

forms correlated well with the presence of the low surface tension of saline extracts. Using whole lung extracts, Harlan et al. (1966)

showed that the phospholipid content was significantly higher in dog and human lungs compared to frog, turtle and chicken lungs. They also showed that pulmonary artery occlusion (a process known to lower

surfactant levels) significantly reduces the phospholipid fraction in whole lung extracts.

The Lung Lining in Various Pathological Conditions

1. Oxygen toxicity has been shown to alter the lung lining

in certain species, but there is conflicting evidence as to whether

breathing oxygen effects all species uniformly. Morgan et al. (1965)

exposed dogs to 100 percent oxygen at one atmosphere for about h8

hours and found by the lavage technique of surfactant removal that

the oxygen-treated dogs had significantly higher minimum surface tensions and a significantly lower phospholipid oontent. Collier

et al. (1965) studied oxygen toxicity in rabbits kept at one atmos­

phere oxygen from 72 to 96 hours; minced lung was saline extracted.

They found a higher minimum surface tension in oxygen-poisoned

rabbits, and bubbles that were less stable. Pattle (1961) exposed

three mice to one atmosphere of oxygen and reported rx> change in

bubble stability. Fujiwara et al. (1964) exposed guinea pigs to

100 percent oxygen at one atmosphere and found no change in minimum

surface tension; they also reported no change in the lecithin or

phospholipid content of the lung. Giammona, Kemer, and Bondurant 13

(1965) compared several species on the basis of altered surface

activity due to oxygen toxicity at one atmosphere. They used both mincing and lavage techniques; cats, rabbits, and rats were com­

pared. An increase in minimum surface tension was reported in cats

and rabbits both by mincing and lavage techniques. In rats the

increased surface tension was found to be the result of the use of

the mincing technique; the lavage technique indicated no change.

It is thus pointed out that the method of extraction is of signi­

ficance.

Three recent studies indicate that the exposure of rats to

hyperbaric oxygen raises the minimum surface tension of lung ex­

tracts. The mincing technique was used in each case (Webb et al..

1966; Jamieson and Brenk, 19^4; Kennedy, 1966). Bondurant and Smith

(1962) using the transudation technique on rats reported that hyper­

baric oxygen at 8 atmospheres for 20 to 45 minutes A.d not alter

the minimum surface tension.

2. The respiratory distress syndrome of the newborn has been

found by most investigators to be related to an increased minimum

surface tension and a reduced lining complex. At autopsy, atelectasis

is found; histology often but by no means always shows a deposit

known as hyaline membrane. The importance of surface tension in the

newborn was stressed by Gruenwald (1947). He also noted as a feature

of the disease (1958) distended respiratory bronchials and collapsed

alveoli; this condition, he states, "is caused by surface tension,

and may be experimentally produced when the lungs are inflated with 14 air but not with fluid.” It was suggested by Pattle (1958) that a deficiency of lining complex might be involved with the causation of the disease. This has since been confirmed by Avery and Mead (1959) t

Pattle et al. (1962) and Clements et al. (1961). More recent evidence tends to contradict this theory (Chu, Clements, Cotton and Klaus,

1965), showing that pulmonary hypoperfusion is the central issue.

3. A considerable range of other pathological conditions have been subjected to various surfactant-surface tension tests. Post mortem changes may present serious changes such as additional col­ lapse of healthy regions and thus obscure any pathological collapse.

Pattle (1961) has determined the stability ratios of extracts from lungs under a variety of pathological conditions. He reports finding no abnormality in any naturally aerated portion of a lung even if part of it showed pathological collapse. He found the capacity of the lung to form a lining film to be unaffected in the collapsed areas which were caused by bronchial obstruction, blast, Indirect injury, and a wide variety of irritants such as phosgene, cadmium oxide, oxygen, blade smoke, and others. Schaeffer et al. (1964) reported that the inhalation of 15 percent OO2 decreased lamellar body formation and raised the minimum surface tension of guinea pig extracts. Findley, Tooley, Swenson, Gardner and Clements (1964) demonstrated that pulmonary hypoperfusion produced by clamping the pulmonary artery caused an increase in the minimum surface tension of lung. Rosenberg et al. (1962) showed with air and saline pressure- volume curves that the force due to surface tension decreased after 15 the inhalation of aluminum oxide particles by rats. It appeared that a better surface active complex was formed.

Mechanical Properties of Lungs Related to Surface Tension

Mechanical Behavior of the Lungs under Nearly Static Conditions

Donders was the first to point out that lungs collapse on opening the chest wall because of their own elastic retraction and that this retractive force (measured in terms of airway pressure) increased as the lungs are inflated (Mead, 1961). At approximately the same time Hutchinson also published results obtained on two human lungs immediately post mortem. His values furnish the earliest pressure-volume curves for lungs. The limited linear relationship went unnoticed (Mead, 1961). Sixty-five years later Cloetta ob­ tained similar results on the lungs of dogs, cats, rabbits, and monkeys. By lowering pressure around the lung, he concluded, there­ fore, that there was a linear relationship between pressure and volume. Setnikar (1955) presented the fallacies in these con­ clusions. The apparent linearity depended on the use of inflation curves over a limited range. Heynsius used Donder’s methods to measure retractive pressures at various volumes. He noted that the number of alveoli open at a given volume and time may effect the results. Liebermeister published pressure-volume ourves from excised lobes from cat and human lungs (Mead, 1961 ). He noted little change in volume as pressure increased from the collapsed state until 8 to 10 cm. 16

HgO pressure was reached at which point the lungs began to fill markedly. None of these workers recorded deflation curves, and consequently did not observe the characteristic hysteresis loop.

More recent results obtained by Radford (1957) and Mellroy (1952) clearly demonstrate this hysteresis. Although Neergaard in 1929 first began to study the role of surface forces on the lung, he used deflation curves only and thus did not observe the hysteresis which was later found to be primarily accountable to surface forces (Mead,

1961).

Surface Tension and Pressure- Volume Curves

Since Neergaard's work was discussed previously it will be mentioned now only where it applies to the review of more recent work.

Mead (1961) stated "The surface and tissue elements containing the surface may be thought of as operating in series. That is to say, the net pressure developed by the surface and the tissue is the sum of separate surface and tissue pressures at any volume." The follow­ ing equation was used by Cook, Mead et al. (1959) to relate surface and tissue compliances:

unit = ^/Cgurface + V^ti ssue

Compliance is defined as the volume change per unit pressure change.

Clements et al. (1958) have said "It is reasonable to suppose that when the alveoli are extended, tissue forces predominate, while at moderate to small volumes, surface forces predominate." Lungs are 17 more stable at large lung volumes. A point of instability can be reached in pressure-volume curves when lungs suddenly open at fairly high pressures. The pressure-volume curve at this point appears to double bade on itself, that is, a place is reached where pressure and volume are inversely related; volume rises rapidly and then a point of stability is again reached (Mead, 1961). As gas free lungs are filled, some areas remain atelectic while others pop open. Filling appears to be uniform during saline filling (Mead, 1961). Qiring air inflation certain alveoli may inflate beyond the hemispheric stage temporarily (the point of instability) and then decrease in volume and regain their stability as the lung oontinues to expand and other alveoli pop open. Surfactant plays a major role in establishing alveolar stability. Recently dements (i9 6 0) reported a high degree of correlation between the volume of air in lungs at maximum infla­ tion and the volume remaining on deflation to 5 cm. water.

There is experimental evidence that most of the hysteresis in gas-filled lungs is due to surface forces. Lungs of dogs, cats, and rats show comparatively little hysteresis when the lungs were filled with liquid (Mead, Whittenberger, and Radford, 1957). When lungs are inflated from the collapsed state, some alveoli which open at low pressure are over-extended before others open to share the load. On deflation, however, more alveoli share the load, all contracting together at the same rate (Mead, 1961). 18

Role of Tissue Forces on Pressure- Volume Curves

Klein was the first to relate retractive pressures of the lung

to the length force characteristics of its tissue elements (Bean,

19^5). He used inter-connected rubber balloons and showed that during

a given filling many different pressures could develop. It was re­

ported that at higher lung volumes pressure rises more slowly, but

that in the case of saline filling the reverse situation exists, that

is, that pressure rises faster as lung volume increases. It was

suggested that collagen fibers are the most probable source of re­

straint.

Dynamic Volume-Pressure Curves and Compliance

Bayliss and Robertson (1939) pointed out in their study of

the visco-elastic properties of cat lungs that at physiological

frequencies gas viscosity could account for only a small part of

the resistance of the total system. Several attempts have been made

to correlate static and dynamic lung compliance. Neergaard (1929)

described the approach which was used to investigate this question.

They let compliance equal tidal volume divided by the change in

pressure from end expiration to end inspiration. Currently, the

term, dynamic compliance, is applied to this kind of measurement.

A number of studies have shown that in most normal lungs dynamic

compliance is independent of breathing frequency up to frequencies

of 60 to 90 breaths per minute (Butler et al.. 1957; Chemiack,

1956; Mead and Whittenberger, 1956; Otis et al.. 1956). 19

Thfl Pulmonary Manifestations of Oxygen Toxicity

Oxygen at One Atmosphere

The first suggestion that breathing oxygen caused pathological effects seems to have come from the observations of Priestly (Bean,

1945). Lavoisier was aware of these observations and having, as he said, occasion to repeat some of Priestley’s experiments chose guinea pigs for the purpose. Autopsies were performed on these animals which had died in "vital air" and he found that in every case death seemed to have been caused by "une fievre ardent" and a "maladie inflammatoire." "The flesh was a very red colour, the heart livid, and turgid with blood, especially the right auricle and ventricle; the lungs were very flaccid, but very red, even externally"; they were also engorged with blood (Bean, 1945). Beddoes, one of the earliest advocates of oxygen for therapeutic use, recognized that oxygen caused extensive pulmonary damage (Bean, 1945). Smith (1899) found pronounced pulmonary damage in mice, birds, rats, guinea pigs and pigeons which had been exposed to oxygen at atmospheric pressure.

The alveoli were filled with a granular exudate; no leukocytes were present. In addition to pulmonary inflammation, congestion, and consolidation, there was also congestion of other organs. Karsner

(1916) stated that "atmospheres containing 80 to 96$ oxygen under normal barometric pressure produce in 24 hours, or more commonly 48 hours, congestion, edema, epithelial degeneration and desquemation, fibrin formation, and finally pneumonia." 20

The French investigator, Paul Bert (1878), was the first to show that a high oxygen concentration can kill many forms of organisms.

Binet and Bouchet observed that guinea pigs exposed to oxygen in con­ centrations of 96 to 98 percent at first tolerated oxygen without much change, but they reported a tendency towards lethargy with alternating periods of activity; the torpor was accentuated, anorexia was common, and the animal curled up in a ball; respiration became dyspneic, and the animal soon died of asphyxia (Bean, 19^5). The lungs showed desquamation, congestion, thickened alveolar walls, leucocytic and eosinophilic infiltration, and generalized congestion. Bean (19^5) concludes that the more outstanding characteristics of oxygen toxicity are; inflammation, congestion, edema, atelectasis, fibrin formation and consolidation in the lungs, pneumonia of various types, bronchitis, hypertrophy, hyperplasia, desquamation and degenerative changes of alveolar cells; sclerotic changes with narrowing, thickening, and hyalinization of pulmonary arterioles, dilatation of the right or both sides of the heart; cardiac hypertrophy and cloudy swelling; congestion of abdominal viscera with cloudy swelling in the kidneys; splenic contraction and testicular degeneration.

Roth (19&J-) has more recently reviewed the effects of high oxygen tensions in animals. He divided oxygen toxicity into two classes according to the target organ hit; at less than two atmos­ pheres the respiratory tract is hit; at greater than two atmospheres the central nervous system is the principal organ affected. Becker-

Freyseng (1939) exposed 50 assorted animals to .80 to .87 atmospheres 21 oxygen for seven days and found severe pulmonary edema, mediastinal

edema, and pleural exudates. Cats and rabbits showed marginal emphy­

sema. Lungs were hyperemic; alveoli were edematous, filled with red and white blood cells, and lined with a debris-filled membrane

(perhaps a "hyaline membrane"). This membrane adhered to vascular walls, extended into bronchioles, and appeared fibrinous in nature.

Penrod (195&, 1959) pointed out that rats and guinea pigs have

endemic lung diseases that complicate pathological studies. He found in cats that by cannulating one bronchus and occluding the other dur­ ing the administration of 100 percent oxygen at several atmospheres

for three hours, he could produce the normally described types of pathology in the open lung but not in the occluded lung. He suggested

that this experiment ruled out the possibility of a blood-borne carrier

as the causative mechanism of pulmonary pathology and that oxygen directly effected the alveoli. Both lungs, however, became atelectatic.

A recent study by Weir et al. (19&1) confirms their findings. Ceder-

gren et al. (1959) showed with the electron microscope that the lungs of mice e^osed to one atmosphere of oxygen for three to six days were changed considerably. There was a patchy thickening of the alveolar wall due either to hypertrophy or fluid accumulation in the cells. The splitting of basement membrane and fluid vacuoles between endothelial cells and membrane were also seen. Roth (196^4-) suggested that this damage was probably responsible for the passage of fluid from blood into the alveoli. METHODS

General Procedure

Pressure-volume curves, compliance values, and lung lecithin determinations were used to study the effects of oxygen toxicity.

Air and saline pressure-volume curves were run on the lungs removed from rats exposed to 99 t 1$ oxygen for 60 to 66 hours. The lungs were filled and emptied by means of a continuous infusion pump. The following sequence of curves was run on each rat: air curve, saline curve, lung perfusion to remove surfactant, another air curve and another saline curve. The lungs were filled to a pressure of 20 cm. water with air and with saline to that volume attained in the air inflation. The perfusion and surfactant washout was accomplished by infusing saline into the pulmonary veins and collecting fluid from the trachea. The lecithin component of this tracheal transudate was determined by thin layer chromatography and used as an index of surfactant. Total lung compliance was determined at the point of maximum inflation by dividing the volume by the pressure at that point. This was done in control and oxygen exposed rat lungs both before and after perfusion. Tissue compliance, based on the saline curves, was determined in each case. That component of compliance due to surface forces, surface compliance, was calculated by means of the following formula (Cbok, Mead et al.. 1959):

^/^total = V^tissue + V^surface

22 23

By these methods the relationship of surfactant to the respiratory difficulties encountered in oxygen-poisoned lungs were analyzed.

Animals

Rats (Wistar strain) were chosen as the experimental animal primarily because of their convenience and availability. Rats have been used extensively for oxygen toxicity studies. Rats between

180 and 260 grams were used. Younger rats, less than 125 grams, have been shown to have prolonged life spans in 100$ oxygen; older rats, weighing more than 300 grams, generally have shorter life spans in

100$ oxygen presumably due to a decrease in their natural resistance to toxic and other pathologic conditions. Although rats have been

shown to have endemic bronchial pneumonia, the diseased lungs were identified by their spotted appearance and discarded. Because of the ease of handling, the rat was a good choice for an experimental animal.

Oxygen Exposure

Rats were exposed to one atmosphere of oxygen for 60 to 66 hours in a polyvinyl chamber (Figure 1). Ihese chambers, 5 feet by

4 feet by 4 feet, were similar to germ-free isolators although no attempt was made in these experiments to maintain sterile conditions.

These isolators were fitted with rubber gloves for easy accessibility to the animals, and ports and gas locks so that a few rats could be put in or removed while still maintaining the nitrogen-free atmosphere.

The isolator was connected to an oxygen tank and was kept inflated Figure 1 Oxygen Chamber 25 with a positive pressure of \ to 1 inch water. Additional structural support was given to hold the isolators open during opening of the ports when a nitrogen-free atmosphere was not being maintained. Ex­ cess humidity was removed by continuously circulating gas from the isolator through a refrigerated oondenser. Relative humidity was kept between 50 and 65$. Carbon dioxide was removed by pumping the gas through cannisters containing soda lime. Samples of gas from the isolator were analyzed daily for percentages of oxygen, carbon dioxide, and sometimes nitrogen. Oxygen was analyzed by the Beckman

E-2 paramagnetic analyzer; carbon dioxide by the Beckman LB-1 infra­ red analyzer; nitrogen on a Med-Science Nitralizer; temperature by a mercury thermometer, and relative humidity by a hair hygrometer.

Oxygen was kept between 98 and 100$ and nitrogen less than 2$; carbon dioxide less than -§$. Rats were kept two or three to a plastic cage with a wire top, 12 inches by 6 inches by 6 inches, with adequate food and water at all times. An air-filled isolator was initially kept for control rats, but was abandoned when lungs were found to be the same as when animals were held conventionally.

Removal of Rat Lungs

Pressure-volume curves were run on the excised lungs of all rats exposed to 100$ oxygen and on corresponding controls of the same weight. The rats were given a lethal dose of sodium pentobarbital and the thorax opened before breathing movements ceased. Those rats which were exposed to oxygen were injected with pentobarbital in air 26 immediately after removal from the isolator. The lungs and trachea were removed as quickly as possible in order to avoid any sudden pulmonary edema which might occur as a consequence of stoppage of the left heart first due to the anesthetic. lung and trachea removal was a delicate process requiring considerable care to avoid leaks.

All leakers were discarded. The leakers were identified by their abnormal pressure-volume curves; the recorded volume did not return to zero after complete deflation as was the case with normal lungs.

After the lung and trachea was removed the trachea was cannulated for the purpose of attachment of the pressure-volume apparatus. At this point in the process the lungs were weighed, and the buoyancy in saline was determined.

Pros sure-Volume Apparatus; Air Iniision

The pressure-volume apparatus in the final stage of develop­ ment worked as follows: the lungs with tracheal cannula in place were suspended in a 60 milliliter rubber-stoppered jar (Figure 2).

A connection was made through the rubber stopper from the trachea to the pressure source — an automatic infusion syringe (Colson

Constant Flow System, Model 1055)* Another connection was made between the atmosphere around the lungs within the jar and a volume recorder (Med-Science Volume Meter, Model 160). The arrangement was such so that the automatic syringe (Figure 3, *0 infused air into the trachea, and as the lungs expanded, they displaced air around the lungs which was forced out of the jar and into the volume recorder.

Volume Lungs Recorder

Figure 3 Pressure-Volume Apparatus Water Manometer

Volumn Meter Air

Pressure £ Saline Transducer Reversible Automatic i i Stoppered Bottle Syringe Amplifier » > (150 ml) Infusion Pum ps■ > Linear Transformer Trachea tied to Cannula Saline Level (Saline Infusion) , Lungs

Saline Level (Air Infusion) axis

«.BB989.9B9B98a98Q8aagg«r» X-Y Recorder y axis

Block Diagram of Apparatus for Automatic Registration of Pressure- Volumn Curves from Excised Rat Lungs Figure 4 30

Pressure was detected from a connection between the infusion syringe and the trachea by means of a Statham Strain Gauge pressure trans­ ducer (Model P23 B). Thus, as air was infused into the lungs, the pressure within the lungs was picked up by the transducer. The pressure around the lungs remained essentially atmospheric. This was due to the characteristics of the volume recorder, which was connected in such a way so that as the lungs expanded they forced air out of the jar surrounding them and into the volume recorder.

The volume recorder sensed (cartesian diver sensor) a very slight pressure change (less than 1 mm Hg) which turned on a servo mechanism within it. The machine contained a cylinder and a piston; a very slight positive pressure caused the piston mechanically to be moved out of the cylinder, thus accepting air from the jar surrounding the inflating lungs. The piston moved until the pressure was pre­ cisely atmospheric. On deflation of the lungs, the piston moved into the cylinder and air entered the jar around the lungs, thus returning the same air which had entered the recorder. A linear transducer was adapted to the sliding arm of the piston, and the output of the transducer was transmitted to the Y axis of a Mosely

Autograph X-Y recorder. Pressure output from the Statham Strain gauge transducer was connected to the X axis, via a Grass (Model 5B) or Brush amplifier. Thus pressure-volume curves were plotted simultaneously and automatically as the lungs were inflated.

Calibration for the sake of convenience was set so that 1 cm. water pressure (X-axis) equaled 1 cm. on the graph paper, selected 31 with 1 cm. divisions, and volume was either set at 1 or •§• ml per cm., depending on the size of the lungs. In order to avoid errors due to gas expansion of compression, all measurements were made at room temperature.

Saline Infusion

Saline infusion was performed on a set-up slightly modified from that used for air infusion (figure 5)• The syringe and all tubes were filled with saline and made bubble free. The jar con­ taining the lungs was filled with enough saline to cover the lungs, about 3 A filled; as the lungs expanded the air at the top of the bottle was forced out and into the volume recorder the same as with air filling. The levels of the infusion syringe, pressure transducer, and saline level around the lungs was carefully adjusted so that all were approximately equal in order to avoid artefacts from a hydro­ static column of saline. Pressure-volume curves were traced the same as the air curves on the X-Y reoorder.

Residual Air in Lungs

The residual air in lungs was estimated immediately after the lungs were removed from the rat and immediately before saline in­

fusions. The process was as follows: a beam balance sensitive to

0.01 grams (Qial-O-Gram, Ohaus) was slightly modified by removing the pan and attaching in its place a flexible wire. On the end of the wire was attached a weight and a hook to attach the lungs; the end of the wire with the weight and the lungs was immersed in a beaker Lungs

Figure 5 Saline Filled Lungs 33 of saline. The lungs held under water by the weight tended to push upward if they were buoyant and hence decrease the weight of the system. The buoyancy of the lungs was in direct proportion to the residual air they contained and the change in weight of the system was recorded as a measure of the residual air the lungs contained at the time of measurement. Frank (1963) used a procedure similar to this; he estimated the lung tissue specific gravity to be 1.065.

Since physiological saline was used as the immersant (specific weight

- 1.01), it was assumed that the tissue weight and the weight of edema fluid was essentially the same as that of saline. Hence, residual air volume was calculated on the basis of one cubic centimeter per gram weight decrease.

3ecause the saline filled lungs that were used in our results normally contained approximately 0.4 ml air, a check was made to see what effect the air might have on the results. It was found that there was no statistically significant difference in the slopes of those lungs containing a small amount of air and the completely degassed lungs.

Perfusion of Lungs to Remove Surfactant

Bondurant and Miller (1962) successfully worked out a perfu­ sion technique to remove surfactant which we modified slightly.

Their technique consisted of cannulating the rat pulmonary artery

(a tedious process) and infusing saline into the pulmonary vasculature in this manner. The trachea was cannulated and connected to a 34 collection bottle in which a negative pressure of -6 cm. water was maintained. The lungs were then placed in a jar in which pressure was alternately made positive (2 cm. water) and negative ( - 6 cm. water) to simulate respiration and aid in the removal of surfactant. As saline under pressure, 20 cm. water, was infused into the pulmonary artery for approximately 20 minutes, it was subsequently forced across the alveolar walls and out through the trachea (Fig. 6 and 7). The surface lining of the alveoli was broken loose and carried along with the saline. The saline plus surfactant left the lungs as a froth.

The success of the technique for surfactant removal has been carefully substantiated by Bondurant and Miller (1962), and shown to be superior to the mincing, chopping, and homogenation techniques (Pattle, 1965)•

The only modification which was applied in this study was to cannulate the pulmonary vein instead of the artery. This was done because the pulmonary vein can be more easily cannulated in the rat through the left ventricle and atrium.

In order to test the efficiency of surfactant removal, 25 ml samples of saline transudate were collected in series and analyzed for their lecithin content. It was found that the first 25 ml of perfusate contained approximately 60$ of the usual lecithin sample for a pair of lungs, and that additional 25 ml samples in sequence contained

1 5$, 10?6f 10$ and 5$ respectively. Thus, the first 125 ml of per­ fusate contained almost all of the lecithin. An additional 100 ml of perfusate was collected as one sample, but there was so little lecithin present in it that it could not be resolved on a Figure 6 Perfusion Apparatus Figure 7

Saline Pressure Bottle 20 cm To Pulmonary Vein Vacuum Alternate Pump Pressure+ 2 cm H20 10cm H20 I V acuum -6cm H20 Stoppered Cycle Pressure- Pump Bottle Valve Vacuum Collection of Transudate Lungs Cannula to Pulmonary Vein via Left Ventricle Cannula tied into Trachea Stoppered Side-arm Flask

Block Diagram of Apparatus for Collection of Pulmonary Surfactant via Perfusion of Pulmonary Circulation

as 37

thin layer plate. It was therefore established as a routine to

collect 1 50 ml of perfusate for each lung lecithin analysis.

Pressure-Volume Curves

The general procedure followed with each rat was: first,

after removal of the lungs to run an air pres sure-volume curve; then

a saline curve; after the saline curve, the lungs were removed from

the pres sure-volume apparatus and were perfused to recover the sur­

factant; a second air curve was then run on the lungs, followed by

another saline curve. Thus four complete pressure-volume curves

were run on each rat lung.

Rate of Filling; Maximum Pressures: Volumes

In order to establish guidelines and a standardization among all pressure-volume curves so that the results could later be analyzed

and compared statistically, maximum pressures and volumes were esta­

blished. It was found by trial and error that when lungs were filled

with air beyond approximately 20 to 2^ cm. water pressure, they tended

to develop leaks. Although some lungs remained intact, it was felt

that this selective process could in some way influence the results.

Other Investigators have villed rat lungs to as high as 30 era* water

pressure, and reported on only those lungs which did not leak. It

should be noted that other investigators have consistently made

pressure the dependent variable, whereas we have made volume dependent,

which could in some way account for the higher percentage of leaks 3B which apparently developed with our system when pressures were above

20 cm. water.

It was decided that a maximum pressure of 20 cm. water was adequate to give a fairly large range of expansion and still avoid leaks due to over-inflation. The lungs used for all air pressure- volume curves both before and after perfusion were filled until a pressure of 20 cm. water was reached; that point was determined by the use of a water manometer as a check on the calibration of the i-Y recorder. When this pressure was reached, the automatic syringe was reversed so the pressure within the lung immediately began to fall (Figure 8). The maximum volume reached depended on the char­ acteristics, that is, the compliance (volume change per unit of pressure change) of the individual lung.

The maximum pressure and volume reached when filling lungs with saline was determined somewhat differently. The volume which was attained in air curves was generally reached in saline curves before a pressure of 2 to cm. water was reached. Setting a maximum pressure was a difficult procedure. If the maximum pressure were set the same as with the air curves, the lung volume, because of a lack of surface forces, reached very high values, greater than 1 5 cc, and the lungs consistently leaked. On the other hand, when maximum pressure was set in the range of 2 cm. water, it was found that accuracy was not very high. In this range an error of 1 /h- ran. water pressure, which was common when trying to read the manometer while pressure was changing, amounted to a 12.5# error whereas in the air

40 curve, which extended over a range of 20 cm. water, the error was only

1.25$. In view of these difficulties, it was decided to reverse saline infusion when the same lung volume was reached as was attained in the previous air curve at 20 cm. water. Indirectly pressure was still the determinant. The pressure of 20 cm. water determined the volume of the air curve which in turn was used as the volume of the saline curve. Thus both air and saline pres sure-volume curves were related to an alveolar air pressure of 20 cm. water.

The rate of filling was set very low in terms of the normal rate of respiration of the rat in order to avoid any significant air resistance or tissue inertia forces. The normal rate of respira­ tion in the rat was shown to be between 50 and 60 breaths per minute

(Rhoades, 1966). The time required to complete one average air pressure-volume curve was 20 seconds or at the rate of 3 cycles

(breaths) per minute. The actual rate of syringe infusion was 16 cc per minute. If 10 seconds (half of the time of an average curve) were allowed for infusion, it can be seen that time would be avail­ able for 2.7 cc of air to enter the lungs. It can be seen in the results that the average lung volume at 20 cm. water pressure is

1.2 cc in oxygen exposed rats and 3.0 cc in control rats. The fill­ ing time is thus dependent on the lung volume at 20 cm. water pressure.

Saline was infused at a slower rate because of the higher viscosity of liquid compared to air. The infusion rate was set at 0.66 cc per minute. The average curve required about 6 to 8 minutes. In order to avoid artefacts on the first filling, all air pressure-volume curves were run six times. The first four curves were not recorded. Curves 5 and 6 appear on each graph in order to

show that by this time a point of stability had been reached.

Perfusate Surfactant Measurements

The perfusate which was recovered from rat lungs after the first set of air and saline pressure-volume curves, was used for a measuremat of surfactant. It should be noted here that when the first saline pres sure-volume curve was run, the saline removed from the lungs contained some of the surface lining complex and that this saline was accordingly added to the subsequently collected perfusate.

The lecithin present In the combined fluid was used as an index of surfactant activity. The validity of this relationship is argued in the Discussion. The procedure in general terms consisted of drying down the perfusate from the original 150 cc to about 10 cc in a flash evaporator, extracting the lipids with chloroform-methanol in a separatory funnel, separating the lecithin component by means of thin layer chromatography, and quantitating the lecithin by the sulfuric acid-dichromate technique. Lecithin values for normal and oxygen exposed rat lungs were then compared. Each of the above mentioned techniques will be subsequently described in detail.

Lecithin Extraction Procedure

The perfusate, approximately 150 cc, was either stored under nitrogen and refrigerated for not more than a week before extraction, 42 or dried down at once and stored in chloroform-methanol for not more than two weeks before extraction. The drying process consisted of plaoing the 150 cc of perfusate in a flash evaporator and allowing to dry under vacuum (less than 1 mm Hg pressure) to an approximate volume of 10 cc. The drying process required about one hour; the temperature of the water bath and the perfusate was kept below 48 degrees centigrade. This was done to prevent oxidation of the lipid

(Folch et al.. 1957)* After drying, the 10 cc of concentrated ex­ tract was centrifuged at 1500 rpm for 10 minutes to remove mucous and cellular debris (Pattle, 19^5) • ^ the centrifugation step was eliminated, lecithin values were found to be very erratic. The following steps were then taken:

1. The 10 cc of concentrated perfusate was placed in a separatory funnel with 10 cc chloroform-methanol 2:1 and shaken for one to two minutes.

2. After standing for three to five minutes, the lower layer including any in ter facial fluff was drained into a 5 0 cc centrifuge tube and centrifuged for five minutes. (The lower layer was pri­ marily chloroform and lipid, the upper layer primarily methanol and water; the interfacial fluff also was primarily chloroform with some methanol and water (Folch, 1957).)

The process of centrifugation was incorporated into Folch* s technique in order to more completely eliminate the interfacial fluff.

The fluff is assumed due to the fact that saline was used instead of water as the third component in the chloroform-methanol mixture. 43

The salt concentration was high because of the drying-down pro­ cess.

3. After centrifugation, the upper layer was removed by means of a long needled syringe and placed in the separatory funnel which contained the upper layer from the previous separation; 5 cc chloroform-methanol were added and shaken for one to two minutes.

Steps one to three were repeated twice.

4. After the third centrifugation, the lower half of the lower layer in the centrifuge tube containing chloroform and lipid was removed by means of a long needle syringe. The upper half was discarded because of some contamination from the fluff layer. The fluff interfered with TLC spotting procedures by stopping up the pipettes.

5. The lower layer, about 1 5 cc of chloroform and lipid, was dried under a stream of nitrogen while being heated (not over k8 de­ grees centigrade) in crucibles placed on a hot plate (temperature block) for approximately 20 minutes. The lipid which formed as a brown spot on the bottom of the cricuble was then stored in a vacuum dessicator until prepared for chromatography.

Thin Layer Chromatography — Separation of Lecithin

Quite a large number of solvent systems and types of silica gel were tried before arriving at a single acceptable technique. It was finally decided that the best method was the use of a solvent system developed by Mangold (1961), which consisted of chloroform- methanol-water 65:25:^. Solvents were reagent grade but were not

redistilled. Plates were of two types. When this processes first

started, plates were prepared by hand, the gel being spread by means

of a glass rod. Later when improved Kodak pre-prepared flexible

plates were available, these were substituted with better success

because of the evenness of the coating. Plates were run for one hour,

dried, the spots located with iodine spray, marked, and heated at

100 degrees centigrade for one hour to remove all traces of iodine.

The spots were then scraped off and the process of quantitation of

the lecithin begun. The details of the above generalization are as

follows:

I. Preparation of Plates

1. High purity silica gel (Brinkman Silica Gel H) was

mixed with distilled water in an approximate 1:2

ratio on a weight basis, shaken thoroughly, and

immediately used.

2. Plates (20 x 20 cm. glass from Brinkman) were thor­

oughly washed and rinsed in distilled water and

ethanol and air dried. They were then laid on a

dean desk blotter and the edges of two sides were

taped to the blotter with masking tape which was

125 microns thick. The tape was applied in such a

way so as to make a ridge along two sides of the

plate. A glass rod could then be passed along the

two ridges without actually contacting the plate. 45

The glass rod thus served as a spreader. The thick­

ness of the tape determined the thickness of layer

of silica gel which was spread. Two layers of tape

gave an approximate thickness of 250 microns, the

ideal thickness for thin layer chromatography.

3. Spreading of the prepared gel consisted of pouring

a mixture of 15 grams of gel and 30 cc water onto one

end of the plate and immediately spreading it across

the plate with the clean glass rod. Reasonably good

plates could be consistently produced with practice.

4. Plates were then stored in a desiccator until used

at which time they were activated by heating them at

100 degrees centigrade for one hour.

5. Kodak pre-prepared flexible plates were ready for use

except for the one hour of activation.

II. Splotting of Sanple and Identification of Spots

1. The sample which had been previously dried in a 15 ml

crucible and stored in a vacuum desiccator was made up

to 0.04 ml with propanol using a Hamilton micro­

syringe.

2. After mixing gently for 5 minutes, 0.01 ml was taken

up in a disposable pipette (Drummel Micro caps) and

spotted between 1 and 2 cm. from the bottom of a

prepared plate. Two spots were made for each sample. i*6

Each plate contained 2 unknowns, 2 standards, and a

blank lane.

3. After drying for 15 minutes, the plates were placed

in a glass chromatography tank containing the develop­

ing solution.

h. After running for one hour, the plates were removed

and let dry for 15 minutes. The plates were then

sprayed lightly with a 6 , 7 . % solution of iodine in

methanol. The iodine served to make the spots visible.

The lecithin spots were immediately circled with a

dissecting needle and the plates were then heated at

100 degrees centigrade for one hour to remove all

traces of iodine, so that it would not interfere with

quantitation.

III. Chromatography Tanks

1. Plates coated in the laboratory were placed on end

in anatomy type glass tanks. The ehloroform-methanol-

water mixture was put in the bottom of the tank at

least 6 hours ahead of time for equilibration to

occur. To help insure a saturated atmosphere, the

walls of the tanks were lined with filter paper.

2. Pre-prepared (Kodak) plates were placed in the more

recent sandwich type tanks where the tank volume is

extremely small and equilibration is rapid. Quantitation of Lecithin

General Procedure

1. Amenta (19&0 modified for the purposes of thin layer

chromatography a quantitation technique developed by

Bragdon (1951) which would be useful for all lipid

classes based on the ability of lipids to reduce an

acid dichromate solution. Amenta found that certain

silica gels did not react with acid dichromate which

made elution unnecessary. The silica gel at the site

of the spot to be quantitated was scraped off the

plate and put directly into the acid dichromate

solution.

2. The acid dichromate solution was prepared by dis­

solving 2.5 grams of K^Crjp-p in one liter 36 normal

H2S0Zf. Two ml of reagent were used for each deter­

mination which allowed adequate reduction for accurate

measurement when the amount of lipid was less than

200 micrograms.

3. An improvement of the relative insensitivity of the

method developed by Bragdon was that of determining

the amount of oxidized CrgO?" remaining, rather than

the amount of reduced Cr3+- produced (Rosen, 1961).

Standard curves were established for lecithin.

Chromatographically pure egg lecithin was chosen

as a standard because egg lecithin was shown by *»8

Pattle and Thomas (1961) to be qualitatively identical

by infrared spectrophotometry to surfactant.

B. Specific Techniques

1. A small amount of silica gel containing the spot to

be quantitated was scraped off the plate with a Clean

razor blade and placed into a 12 ml glass-stoppered

pyrex centrifuge tube into which had previously been

placed 2 ml sulfuric acid dichromate solution (pre­

pared according to Amenta*s technique as described

above).

2. The mixture was shaken and heated in a dry block

heater at 100 degrees centigrade for 45 minutes. It

was shaken twice during the heating.

3. The solution was then cooled and centrifuged to

separate the silica gel.

4. When pro-prepared plates were used, the process was

slightly modified. The gel on the pre-prepared plates

did react with the sulfuric acid-dichromate reagent

but by eluting with 10 ml chloroform-methanol 2s1,

the lecithin was extracted from the gel. The extract

was dried down under a stream of nitrogen, then the

sulfuric acid dichromate reagent added, and the pro­

cess continued the same as above. After heating for

45 minutes, the reaction was complete. One-half ml of reacted mixture was removed from each tube and placed in a 20 ml cuvette and 1 ^ ml dis­ tilled water added.

After mixing, the absorbance was determined on a

Bausch and Lomb Spectronic 20 spectrophotometer. The absorbance was determined at 350 mu according to

Amenta's technique.

The difference in absorbance was determined between the sample or standard and blank containing unreduced sulfuric acid-dichromate solution. A gel blank was run in each case to eliminate oxidation due to im­ purities in the silica gel as a source of error.

The calculation of the results was based on a standard curve made using egg lecithin in 25 microgram incre­ ments. A standard, run with each unknown, was com­ pared with the standard curve for a check on accuracy.

The actual reading, based on a comparison with the

standard curve, was multiplied by a factor of eight,

A factor of 8 was used because the original sanple was halved three times. Once after separation only half of the sample was picked up to avoid all of the inter­ facial fluff. Only half the sample was used after

elution to avoid all of the precipitated silica gel.

Usually two spots were plated, half of the remaining

sample in each spot. 50

Compliance Determination and Analysis of Data

Air pressure-volume curves were analyzed in terms of volune

at four specific pressures: 5. 10, 15» and 20 cm. water pressure

for both inflation and deflation limbs. The volume at each of these pressures was recorded for control rats and oxygen exposed rats, both before and after perfusion in each case. Compliance values for the

total lung were calculated by dividing the volume at a pressure of

20 cm. water by 20. At this point, the point of maximum inflation,

air flow was momentarily static.

Saline curves were analyzed by determining the inflation and deflation slopes by means of a protractor, with which the angLe was read, and a tangent table for converting angles to slopes. The pro­

cess was followed on controls and oxygen rats both before and after perfusion.

A comparison was made on lung volumes between control and oxygen exposed rats; in addition a comparison was made between before and after perfusion volumes in controls and oxygen animals.

A comparison was also made between air and saline curves by which the compliance of the surface forces could be calculated. The following equation was used (Cook, Mead et al.. 1959):

^/^total = ^/^tissue + ^/^surface

Ctotal was the compliance of the air determined at 20 cm. water pressure and C-t^ssue the compliance of the saline curve. the unknown, was the effect of surface tension on the lungs. 51

All volumes were averaged and standard errors computed, and all comparisons were made with "student1s" t-test. Paired dif­ ferences were used wherever possible to reduce errors in the comparisons (Snedecor, 1956). RESULTS

Pressure-volume (P-V) curves were run on excised rat lungs both before and after perfusion for removal of surfactant. Infla­ tion and deflation limbs were recorded from lungs filled with air and with saline. Normally the lungs were filled with air six times in succession to allow for stabilization of the P-V curve. The first two air curves were markedly different from each other (Fig. 9), the second and third air curves differed less, and curves 5 and 6 were almost identical. This was taken to indicate the attainment of

stability In the pressure-volume relationship, and only curves 5 and 6 were recorded routinely (Fig. 10). The upper two curves in Figure 10 are from a control rat before perfusion, and the lower two curves were recorded after the lung had been perfused with saline. The markedly divergent paths for inflation and deflation, producing the typical hysteresis loop, are clearly evident. In agreement with the concept that perfusion removes surfactant and thus increases surface tension, volumes at any given pressure both on Inflation and deflation

(that is, compliance) decreases following perfusion.

A summary plot indicating the effects of perfusion on the normal air pres sure-volume curve is seen in Figure 11. These curves are based on the average of volumes taken from the individual animal curves at pressures of 0, 5, 10, 15 and 20 cm. water pressure.

52 Six Successive Ar P-V urves on an Excised Rat Lung

Vo I

5 10 15 20 Pressure-cm HoO

Figure 9 Air P-V Curve frorr a Confrol'Rat Before an d j\ lex Perfusion to Remoe Surfaetant-Curves 5&6 hown

Vo I

5 10 15 20 Pressure-cm H20 Ui Figure 10 Summary - Control Pressure - Volume Curre Before and After Perfusion

Before Perfusion After Perfusion

t

Pressure-cm H20 Ui Ln Figure 11 56

Again, the smaller volumes at any given pressure indicates a reduced

compliance following perfusion, A statistical analysis (t-test) of

these results (Table 1) indicates significant decreases of about

50/6, on the order of 1 ml or better, in lung volumes following perfu­

sion at pressures of 5» 10» 15» and 20 cm. water for both the inflation limb and the deflation limb. The "complete sequence" group described in the upper part of Table 1 refers to those rats on which the fol­ lowing chronological sequence of pressure-volume curves were run:

air, saline, perfusion of the lungs, another air curve and a saline

curve. A typical graph of this sequence in seen in Figure 12 (a detailed explanation will be introduced later). For these complete

sequence rats, standard errors and P values for comparing effects of perfusion were based on paired differences (the volume difference at

the given pressure before and after perfusion for each rat).

The data labelled "all rats" in Table 1 refers to group averages and differences in volumes at a pressure of 20 cm. water

(maximum pressure) derived from all normal pressure-volume curves regardless of whether or not the complete sequence was obtained.

The data from this larger group of 58 rats before perfusion and 29 after perfusion confirms a significant decrease in lung volumes

following perfusion.

Although 112 additional pressure-volume curves were recorded,

they were not used because of either leaks, noted by a failure of

the recorded volume to return to its zero point; or by bronchial pneumonia, noted by a spotted appearance of the lungs grossly; or TABLE 1

STATISTICAL ANALYSIS OF CONTROL P-V CURVES

Before Perfusion After Perfusion Paired Difference Pressure No. Volume No. Volume (cm HoO) Rats (ml)av. S.E. Rats (ml)av. S.E. (Before-After) SEMD1 P value Complete Sequence 5 12 .39 .065 12 0.07 .019 0.3 .020 .01

10 12 1.02 .148 12 0.23 .031 0.8 .146 .01

Inflation 15 12 1.61 .068 12 0.33 .028 1.3 .226 .01

20 12 2.98 .336 12 1.66 .420 1.3 .495 .05

15 12 3.03 • 338 12 1.77 .428 1.3 .480 .05 Deflation 10 12 2.59 .276 12 1.34- .263 1.3 .323 .01

5 12 1.65 .150 12 0.68 .136 0.9 .179 .01 All Rats Group Difference 20 58 3.20 .437 29 1.60 .249 1.6 .05 Compliance ml/cm. S.E. ml/cm. S.E. (Before-After) Complete HoO HgO Sequence 20 12 0.15 .017 12 0.09 .021 .06 .05

All Rats 20 58 0.16 .022 29 0.08 .013 .08 .05

1 SHMD = Standard error mean difference 20 i Pressure-cm H20 , Air Curve i i _ J i- i______

0 12 oo Pressure-cm H20 , Saline Curve Figure 1 2 59 for technical difficulties with the infusion syringes or in calibra­ tion of the X-Y recorder.

In the last two lines of Table 1, compliance values are indi­ cated, as calculated by simple division of the volume in ml at maximum pressure (20 cm. water) by 20. The significance of the change in compliance, consequently, is the same as the change in lung volumes at 20 cm. Perfusion produced significant changes in total lung com­ pliance both in the complete sequence groip (12 rats) and in all rats

(58 before perfusion and 29 after perfusion).

Normal rat lungs were filled with saline incsdiately after the air curve was run both before and after perfusion. The typical saline curve (Fig. 13) bas a much steeper slope than its corresponding air curve. In order that the complete curve could be recorded, saline curves were usually started near the center of the page (Fig. 14), although the air curves were still startedas usual at the left hand margin. It may be noted that the deflation limb of the saline curves generally have a lower slope than inflation, and that it falls off progressively and does not return to zero; approximately f to 1 ml of saline remains in the lungs, as shown by increased lung weight after perfusion (Table 10) and may be similar to edema fluid. The short horizontal segment between the inflation and deflation limbs is thought to be an artefact due to hydrostatic pressure fluctuations in the apparatus so that the indicated hysteresis loop is consider­ ably exaggerated. 12- rsuec H20 Pressure-cm iue 13 Figure Control Air and Sal'ne P-V Curves

Vo I

Safine i i Air

20 Pressure-cm HoO, Air Curve i i j i i 0 I 2 Pressure-cm hLO,Saline Curve Figure 14 62

The analysis of saline curves was based on the angle of the inflation limb and that of the deflation limb. This angle was measured with a protractor and then converted to a slope (that is,

compliance when expressed as ml volume/cm water pressure) by means of a tangent table. This was easily done for the straight inflation limb, but required some interpolation for the curving deflation limb.

The results for the complete sequence lungs are seen in Table 2.

Three saline values were not included in this analysis. It was

found that one value out of 12 before and two out of 1 2 after per­

fusion were greater than 10 times the average value of the group.

On the basis that such a large difference was an artefact, probably

due to mechanical factors, these values were eliminated. It is noted

in Table 2 that perfusion tended to increase the slopes but not

significantly. This is true for both the inflation and deflation limbs but more so for inflation. Cook, Mead et al. (1959) showed

that edema caused similar results with dog lungs, supporting the

idea that perfusion resulted in a certain degree of edema as well

as surfactant wash-out.

Oxygen Poisoned Rats

Rats were exposed to 99 - 1 $ oxygen for 60 to 66 hours and

the same type of analysis carried out on the lungs as that just

reported for control rats. Typical fifth and sixth pressure-volume

curves before perfusion (upper curves) and after perfusion (lower

curves) are shown in Figure 15 for an oxygen poisoned rat. Note TABLE 2

CONTROL SALINE PRESSURE-VOLUME CURVES

Paired Difference Before Perfusion After Perfusion Compliance No. Angle Slope SE No. Angle SLope SE (before- SEMD1 P rats (degrees) Compliance rats (degrees) Compliance after) value (ml/cm (ml/cm HjjO) (ml/cm H^0) CO o 1 • Inflation 9 37 1.9 .156 9 39 2.7 .*11 • NS2

Deflation 9 2k 0.6 .090 10 26 0.8 .339 - 0.2 .103 NS

1S M D = standard error of mean difference

^NS = not significant

O n VjJ from an Oxygen ExposecTRdf 1 ter^ !j: T";;Lj:-J't

Vo I

5 10 15 20 Pressure-cm H20 Figure 15 65

that both curves are considerably flatter (less compliant) and much more similar to each other than was the case of the typical control

curves (Fig. 10). Figure 16 shows on an expanded scale a summary air

curve for oxygen poisoned rat lungs (on complete sequence animals)

before and after perfusion which further demonstrates the flattening of the curves.

A statistical analysis of both the complete sequence groups

and of all rats (handled in the same way as control rats) is given in

Table % The decreases in volume and compliance are generally signi­

ficant but relatively small compared to oontrols (Table 1). At a pressure of 20 cm. water for the oxygen rats on which a complete

sequence was run, the average volume decrease was 0.6 ml (P less than

0.05), but for all rats the decrease was only 0.1 ml which was not

significant. Compliance changes shown at the bottom of Table 3* are

0.03 and 0.01 ml per cm. water respectively for complete sequence and

all rats, compared to 0.6 and 0.8 for controls (Table 1). The saline pressure-volume curves in oxygen poisoned rats (Table h-) are not as

steep as for controls (Table 2) and were not changed by perfusion.

It was found here too, as with the controls, that two values out of

12 were greater than 10 times the average value of the group and

again, on the basis that such a large difference was an artefact, probably due to mechanical factors, these values were eliminated.

A typical graph of saline curves before and after perfusion is seen in Figure 17, along with the air curves. Summary - Oxygen Pressure - Volume Curve Before and After Perfusion

Before Perfusion After Perfusion

Pressure-cm H20

Figure 16 TABLE 3

STATISTICAL ANALYSIS OF OXYGEN P-V CURVES

Before Perfusion After Perfusion Paired Difference Pressure No. Volume No. Volume (cm HoO) Rats (ml)av. S.E. Rats (ml)av. S.E. (Bef ore-After) SEKD1 P value

Complete Sequence 5 12 0.23 .017 12 0.00 .000 .23 .017 .01

Inflation 10 12 0.65 .067 12 O.UO .022 .U8 .079 .01

15 12 0.89 .090 12 0.26 .029 .63 .105 .01

20 12 1.38 .116 12 0.83 .206 .60 .217 .05

15 12 1.39 .109 12 0.86 .239 .53 .230 .05 Deflation 10 12 1.36 .107 12 0.73 .199 .63 .196 .01 OO O Lf-\ 5 12 0.98 • 12 0.53 .139 .44 .129 .01

All Rats Group Difference 20 US 1.9 .191 27 1.8 .279 0.10 NS2 Compliance ml/cm. S.E. ml/cm. S.E. (Before-After) Complete HoO Ho° Sequence 20 12 0.07 .006 12 0.04 .010 .03 .05

All Rats 20 us 0.10 .010 27 0.09 .014 .01 NS

1SEMD ■ Standard error mean difference % S = not significant TABLE 4

OXYGEN SALINE PRESSURE-VOLUME CURVES

Paired Difference Before Perfusion After Perfusion Compliance No. Angle Slope No. Angle Slope (before- SEMD P rats (degrees) Compliance SE rats (degrees) Compliance SE after) value (ml/cm H^) (ml/an HgO) (ml/cm H2O)

Inflation 10 3^ 1.3 .123 10 y* 1.3 .106 0.0 .000 NS2

Deflation 10 13 0.3 .030 10 18 0.^ .03^ 0.2k .100 NS

1 SEMD = standard error of mean difference

2NS a not significant

& i Sequence of P-V Curves -[Oxygen Ex posed Rat — TtTTTTt— r^:r-TT— j— -T^— I—Tj— Before Perfusion—“ • L |l-“^,ir ;2-Saline ; ,!i :iL - _ i 1—| _ After Perfusion.. . i-3-^Air. 14~Saline 1 1---.

Vo I

20 Pressure-cm HQ0 , Air Curve

J I L

0 I 2 O n vO Pressure-cm H20 , Saline Curve Figure 1? 70

Comparison of Normal and Oxygen Poisoned Rats

The flattening of the pressure-volume curves caused by oxygen exposure is shown graphically before perfusion (Fig. 18) and after perfusion (Fig. 19) for the complete sequence rats. The analysis of this data in Table 5-A shows that before perfusion, oxygen toxicity decreased lung volume at all pressures by nearly 50$. These results are sv?)ported by the analysis of the total group of rats (Table 6).

The compliance values, based on the lung volumes at the pressure of

20 cm. water, are of course also significantly decreased before per­ fusion (Table 5-C and 6-B). After surfactant removal, however, the decreases in lung volumes and compliance due to oxygen while still sizable, are not significant (Table 5-3,C and Table 6).

Table 7 compares the volume decreases (paired differences) due to perfusion in the controls (Table 1) to that found in the oxygen exposed rats (Table 3)» It is clearly seen that the decrease in the oxygen animals was less than half that of the controls, although the difference in decrease could only be shown to be significant in two out of seven comparisons. When group differences in all rats are considered (^9 before perfusion and 27 after perfusion) there is a significant change from 1.6 ml in controls to only 0.1 ml in oxygen poisoned rats (Table 7)* It seems reasonable to conclude from this that oxygen poisoning does reduce the changes in lung slope which normally follow surfactant removal. Figure 13

Before Perfusion Air Curves

Control - 12 Rats Oxygen -12 Rats

5 IO 15 20 Pressure-cm H20 Control Oxygen 73

TABLE 5

STATISTICAL ANALYSIS OF AIR CURVES - COMPARISON OF CONTROL AND OXYGEN EXPOSED RATS, COMPLETE SEQUENCE

Control (12 rats) Oxveen (12 rats) Pressure Volume S.E. Volume S.E. P value (cm H^O) (ml) (ml)

A- Before Perfusion

Inflation 5 0.39 .065 0.23 .017 NS1 10 1.02 .148 0.65 .067 .05 15 1.61 .068 0.89 .090 .01 20 2.98 .336 1.38 .116 .01 Deflation 15 3.03 .338 1.39 .109 .01 10 2.59 .276 1.36 .107 .01 5 1.63 .150 0.98 .085 .01

B. After Perfusion

5 0.07 .019 0.00 .000 10 O.23 .031 0.16 .022 NS Inflation 15 0.33 .028 0.26 .029 NS 20 1.66 .4-20 0.83 .206 NS Deflation 15 1.77 .4-28 0.86 .239 NS 10 1.3^ .263 0.73 .199 NS 5 0.68 .136 0.53 .139 NS

C. Compliance Av. S.E. Av. S.E.

Before Perfusion 20 0.15 .017 0.07 .006 .01

After Perfusion 20 0.08 .022 0.04 .010 NS

1NS = not significant I

74

TABLE 6

STATISTICAL ANALYSIS OF AIR CURVES - COMPARISON OF CONTROL AND OXYGEN RATS, ALL RATS

Control Oxyeen P No. Rats Volume at SE No. Rats Volume at SE Value 20 cm H^O 20 cm HgO

P-V Curves

Before Perfusion 58 3 .2 •ky? 49 1 .9 .191 .0 5

After Perfusion 29 1.6 .249 27 1.8 .279 NS1

Compliance Compliance SE Compliance SE

Before Perfusion 58 0.16 .022 49 0.10 .010 .0 5

After Perfusion 29 0.08 .013 27 0 .0 9 .0 1 4 NS

1NS = not significant 75

TABLE 7

STATISTICAL ANALYSIS - CONTROL AND OXIGEN COMPARISON OF PAIRED DIFFERENCES, 12 RATS

Control Oxygen Pressure P value (Before- SEMD (Before- SEMD after) after)

5 0.3 .020 0.2 .017 NS1 Inflation o o • • 10 0.8 .146 3 NS

15 1.3 .226 0.6 .105 .05

20 1.3 .495 0.6 .217 NS

15 1.3 .480 0.5 .230 NS Deflation 10 1.3 .323 0.6 .196 NS u-% 0 5 0.9 .179 0.4 .129 *

All Rats 20 (49 rats) (27 rats)

1.6 — 0.1 .01

1NS = not significant 76

Oxygen poisoning significantly reduced the slopes of saline curves (Table 8) both before and after perfusion. This change indicates that oxygen toxicity significantly decreased the com­ pliance due to tissue forces (tissue compliance), since surface forces should have been eliminated by using liquid to fill the lungs.

Compliance changes due to changes in surface forces (surface compliance) as opposed to those compliance changes due to tissue forces were calculated from the complete sequence of all the pressure- volume curves. Typical graphs on which the complete sequence was run are seen in Figure 12 (controls) and Figure 17 (oxygen poisoned rats). The upper air curves (zero point starts on the left hand border) are before perfusion, and the lower air curves after perfusion.

The saline curve (zero point starts near the center of the page) slightly to the left is before perfusion and that to the right after perfusion. Total compliance (measured from the air curves) is made up of tissue compliance (from the saline curve) and surface com­ pliance, according to the following relationship (Cook, Mead et al..

1959):

^/^total = ^^tissue+ ^/’"'surface

All compliance measurements were made at 20 cm. water pressure in the air curves.

A coiqplete analysis of the effects of oxygen poisoning on the three compliances is given in Table 9. The significant decrease in the total compliance and compliance due to tissue forces is again 77

TABLE 8

STATISTICAL ANALYSIS OF SALINE CURVES

______Control______Oxygen No. Rats Slope SE No. Rats Slope SE P Compliance Compliance Value (ml/cm HgO) (ml/can HjO)

Before Perfusion

Inflation 11 1.8 .150 12 1.3 .120 .05

Deflation 10 0.6 .090 11 0.2 .081 .01

After Perfusion

Inflation 10 2.6 .400 11 1.3 .169 .01

Deflation 12 0.8 .198 11 0.3 .033 .01 78

TABLE 9

SURFACE COMPLIANCE VALUES

Control______Oxygen tfo. Rats Compliance SE No. Rats Compliance SE P (ml/cm) (ml/cm) Value

A. Inflation

Surface 11 0.17 .021 12 0.07 .007 .01

Tissue 11 1.80 .150 12 1.30 .120 .05

Total 12 0.15 .017 12 0.07 .006 .01

B. Deflation

Surface 10 0.21 .027 11 0.10 .036 .05

Tissue 10 0.60 .090 11 0.2*f .081 .01

Total 12 0.15 .017 12 0.07 .006 .01

Before Perfusion After Perfusion No. Rats Compliancei SE No. Rats Compliancei SE

C. Control Deflation

Surface 10 0.21 .027 12 0.11 .032 .05

Tissue 10 0.60 .090 12 0.75 .099 NS1 Total 12 0.15 .017 12 0.09 .021 .05

D. Oxygen Deflation

Surface 11 0.10 .036 11 0.06 .020 NS

Tissue 11 0.30 .018 11 0.32 .033 NS

Total 12 0.07 .008 12 0.0^ .010 .05

1NS = not significant 79 depicted, but in addition it may be seen that compliance due to surface forces is also significantly reduced. This is true whether tissue compliance is calculated from the inflation or deflation limb

(Table 9-A and 9-B, respectively). Also as shown before (Table 2) in control rats surfactant removal by perfusion (Table 9-C) signi­ ficantly decreases the total compliance and slightly increases tissue compliance, but in addition it may now be seen that the compliance due to surface forces is significantly decreased. In oxygen poisoned rats (Table 9-D) removal of surfactant by perfUsion significantly decreases total compliance and does not significantly change tissue compliance, as showi before (Table 3). In addition, however, it is now shown (Table 9-D) that compliance due to surface forces is not significantly changed. (In both the controls and oxygen rats, these calculations shown are based on the deflation limb of the saline curve, but similar conclusions hold for the inflation limb.) It is thus concluded that both oxygen poisoning and perfusion decrease the compliance due to surface forces, but that oxygen poisoning also decreases the compliance due to tissue forces.

The transudate produced by lung perfusion was analyzed for its lecithin content. Lecithin has been shown to be the primary com­ ponent of surfactant and has been used extensively (Pattle, 1965) as an index of surfactant. It was found that there was a greater than 50$ decrease in the lecithin content of the transudates from oxygen poisoned rats. The value changed from 623 micro grams in normal rats to 293 micrograras in oxygen poisoned rats (Table 10-1). 80

TABLE 10

COMPARISONS BETWEEN CONTROL AND OXYGEN EXPOSED RATS OF BODY WEIGHT, LUNG WEIGHT, BUOYANCY, AND LECITHIN

Control Oxygen-exDOsed No. Mean SE No. Mean SE P Rats Value Rats Value Value

A. 3ody weight (grams) 12 218.7 8.7 12 202.8 6.8 NS1

3. Lung weight (grams) 12 2.2 .104 12 3.0 .420 .01 Before perfusion c. Lung weight (grams) 12 4.0 .204 12 4.4 .175 NS After perfusion

D. Lung Buoyancy (grams) 12 0.44 .029 12 .47 .029 NS

E. Lung Lecithin (micro grams) 15 623.3 47.782 14 292.9 34.830 .01

1NS = not significant A correlation analysis between surface compliance and lecithin was run on 10 rats in which both lecithin values were determined and in which a complete sequence of pressure-volume curves was obtained.

(Most of the 29 lecithin determinations reported were not done on the same rats on which the complete series of pressure-volume curves were run, but on some of the other rats in the larger total group. On the

24 rats on which the complete pressure-volume series was run only a part of the lecithin determinations were successful - success being based on a reasonable agreement between two to three thin layer chromatographic determinations on the same sample.) For these 10 animals, a highly significant correlation of 0.82 between surface compliance and lung lecithin was found (Figure 20). The grouping of the four points from the oxygen animals at the lower values and of the six points from controls at the higher values is clearly seen.

Table 10 also gives comparisons between the complete sequence control and oxygen animals on body weight, lung weight, lung buoy­ ancy, and areas of pressure-volume curves. There is no significant difference in body weight between the control and oxygen groups. The significantly larger lung weight in oxygen rats (36#) is probably due to edema fluid. Lung buoyancy (an indication of residual air in the lungs immediately after removal from the rat) was not affected by oxygen toxicity. The areas of the air pressure-volume curves (area within inflation and deflation limbs) was significantly smaller in the oxygen animals before but not after perfusion. i i

Compliance Due to Surface Forces 0.1 0.2 0.3 200 30 ± 07 .0 0 ± 0 .3 0 30X0.03 + 0 .3 0 82 .8 0 ufc Compliance-Lecithin Surface Figure20 Lecithin (Micrograms) 0 0 4

Oxygen A ControlO 0 0 6

0 0 8 8 3

Grossly normal lungs appeared pink and aerated -whereas the lungs of oxygen poisoned rats were dark colored in areas. Red areas apparently indicating some degree of atelectasis were noticeable on most poisoned lungs. A few lungs were completely liver-like in appearance and did not float. On opening the thoracic cavity most of the lungs of the oxygen exposed rats were surrounded by a clear mediastinal fluid. The amount of fluid varied from about 2 to 8 ml although it did not correlate with any of our other values. DISCUSSION

General

Among the observations made in this study •which lend them­ selves to further analysis and discussion are the following:

1. The partition of total compliance of the excised rat lung

into a component due to tissue elasticity and one due to

surface forces, via the use of pressure-volume diagrams

made by inflating the lungs with air and saline. Such

techniques have been suggested (Cook, Mead et a l . . 1959)

but not used before.

2. The decrease in total lung compliance of the normal rat

lung which followed perfusion of the pulmonary circulation.

This presumably is due to surfactant washout, but has not

been demonstrated before.

3. The relatively small change in the tissue component of

total lung compliance which followed perfusion of the

lungs, despite the fact that such lungs retained some

fluid (were edematous). Somewhat related results have

been reported for the dog, but have not been shown in

the rat.

4. The effect of oxygen toxicity in decreasing lung com­

pliance of the rat both by increasing tissue rigidity and

84 8 5

increasing surface forces. Previous studies had indi­

cated little effect of oxygen toxicity on the surface

forces of the rat lung (Giammona et al . . 1966).

5. The decrease in surfactant in the oxygen poisoned lung,

as extracted by the perfusion technique and quantitated

by separation of the lecithin component of the perfusate

on thin layer chromatography. Previous work with the

oxygen poisoned rat using the mincing technique had

produced no apparent change in surfactant.

6. The good correlation between the amount of surfactant

extracted from rat lungs (both normal and oxygen exposed),

and the decrease in surface compliance which followed the

extraction. This technique appears not to have been used

before, but would seem to be useful in examining the

relationship between surfactant and compliance.

Effects of Oxygen Toxicity on Pulmonary Surfactant

There appears to be general agreement that exposure to greater than one atmosphere oxygen for prolonged periods of time results in a decrease in pulmonary surfactant. At one atmosphere of oxygen there is less agreement on the decrease in surfactant in certain species, especially in rats. Part of this dispute is probably due to different techniques of surfactant extraction.

Several extraction techniques have been used: 1) mincing and chopping, 2) homogenization, 3)lavage (foam fractionation), k) 86 perfusion-transudation. Webb et al. (1966) using mincing techniques

and the Wilhelmy balance reported that three atmospheres of oxygen pressure for from one to six hours significantly raised minimum

surface tensions in guinea pig3 and rats. The animals' lungs were

extracted immediately after death. Jamieson and van den Brenk (1964-)

exposed rats to five atmospheres of oxygen until breathing ceased,

extracted surfactant by the mincing method, and found the raised minimum surface tensions indicating a decrease in surfactant. Kennedy

(1966) exposed rats to two atmospheres of oxygen for 17 to 18 hours, minced the excised lungs, and reported that surface tension was

abnormal in 80 percent of the exposed rats. Contrary to the usual

findings, Bondurant and Smith (1962) exposed rats to eight atmospheres

of oxygen for 20 to 45 minutes, extracted by the saline perfusion

transudation technique, and found that oxygen intoxication produced

no effect on pulmonary surfactant. It is possible that such an

extremely short exposure to oxygen did not allow sufficient time

for any alterations in surfactant to occur.

Several papers have also recently appeared on the effects of

exposure to one atmosphere of oxygen. Those results which are in

most disagreement with our own were reported by Giammona, Kerner,

and Bondurant (1965). They compared the effect of exposure to one

atmosphere of oxygen in cats, rabbits, and rats. Their surface

tension measurements were made on a Wilhelmy balance. They extracted

surfactant by both mincing and by the foam fractionation (lavage)

technique and compared the results using these two methods in each 8? species. They reported that in both cats and rabbits minimum surface tension was higher (less surfactant was present) in animals exposed to oxygen when both mincing and lavage techniques were employed.

However, rats did not react in this way. The results of surface tension measurements on rats exposed to oxygen when the mincing technique was used indicated higher minimum surface tensions, but using the lavage method, the same surface tensions were found in oxygen exposed rats as in control rats. They concluded that,

Minced lung extracts may have abnormally high surface tension in the OIAP (oxygen intoxication at atmospheric pressure) rats because there is not enough air-liquid intermixing to release the surfactant or because com­ parable weights of lungs from normal and oxygen intoxi­ cated animals may not contain comparable amounts of surfactant.

The first of these objections, to mincing because of inadequate air- liquid intermixing, was met in our procedure by using in place of mincing the perfusion transudation technique which according to

Bondurant and Miller (1962) removes surfactant significantly better than other procedures so far employed. The surfactant comes out the trachea as a foam thoroughly mixed with air. The second objection, due to comparing lung of different weights (oxygen lungs are heavier due to edema fluid, and if the same amount of surfactant were present in both control and the oxygen lungs, control lungs would appear to have more surfactant on a per gram lung wet weight basis) was met by comparing lecithin values on a per lung basis, thus eliminating the increase in lung weight due to edema fluid. Either way the comparison is made, our results consistently indicated a highly significant decrease in lung lecithin in rats exposed to 100 percent oxygen. 88

There are two additional sources of difference between the results of Giammona et al» and ours. One was that the animals were kept in oxygen until death, which consistently results in more com­ plete alveolar collapse and thus, according to Levine and Johnson

(1964) greatly inhibits extraction by the mincing technique. The other difference was that of air drying of the extracts. According to Pattle (1965):

A fourth class of test uses derivatives of lung washings, such as air-dried perfusates. In view of the known peculiar chemical properties of lipoproteins, the re­ viewer would be unwilling to draw any conclusions at all from this class of experiment.

ALthough Pattle highly criticizes mincing and chopping techniques, he said, "Most of the objections to extracts of chopped or minced lung do not apply to washings obtained by perfusion of the pulmonary cir­ culation and recovery of washings from the trachea, as described by

Bondurant and Miller (1962)," (the technique used in our procedure).

Other papers have appeared on the effects of one atmosphere of oxygen on pulmonary surfactant. Fuji war a et al. (1964) exposed guinea pigs to one atmosphere of oxygen for various durations of time, extracted surfactant by the mincing technique, and concluded that oxygen toxicity did not effect the pulmonary surfactant in the guinea pig. In spite of the development of severe pulmonary edema, they found no changes in either the surface activity of the extract or in the lipid content of the saline extract of the lung mince. Giammona,

Kerner, and Bondurant (1965) convincingly demonstrated that mincing as used by Fujiwara is not an acceptable procedure for the removal 89 of surfactant in the case of oxygen toxicity. This belief was backed up by Pattle (1965).

Morgan et al. (1965) exposed eight dogs to one atmosphere of oxygen from W- to 52 hours. They reported that although all eight dogs developed respiratory distress symptoms - restlessness, saliva­ tion, labored respiration, and lethargy - only three of the dogs also developed pulmonary edema. All results were compared to only two control dogs; the lavage technique of extraction was used. In the five dogs which developed respiratory distress without pulmonary edema, minimum surface tensions were reported to be only slightly higher than in control dogs, and the total lipid distribution was essentially normal. The fatty acid, palmitate, was significantly reduced in the lecithin fraction, even though lecithin, expressed as a percent of total lipid, did not change. In the three dogs which developed pulmonary edema, surface tension values were slightly higher than in controls, the percent phospholipids decreased, the percent of lecithin remained about the same as control values, and as in the other dogs, the palmitate levels decreased to about one- third of the control values.

Based on the authors' summary as outlined above, it would appear that these findings upset the theories drawn from other results. However, the small number of animals (only two controls were cited) and the very wide range of values make the validity of the conclusions somewhat questionable. The values for minimum surface tension were given as follows: controls - 1 3 and 20 dynes 90 per cm., oxygen-exposed dogs - 6, 7, 25, 22, 16, 23, 22, 30 dynes per cm. Lung lipid values also were scattered over a wide range, total lipids running from 19 to 220 mg in oxygen exposed dogs; of those dogs with edema, two were in the 200 to 250 mg range whereas all the rest (six) of the dogs were in the range from 19 to ^5 mg. Since other lipid values are expressed as a percentage of total lipid, their meaning also is not certain; a statistical analysis was not given.

Caldwell, Giammona, and Bondurant (1963) analyzed the lung extracts of dogs placed in one atmosphere of oxygen. The right lung of each dog was minced and the left lung perfused and the surfactant removed by the transudation technique. It was shown that both tech­ niques resulted in a statistically significant increase in minimum surface tension in oxygen exposed dogs. Their results showed minimum surface tension levels in control dogs to be 2 ± 8 dynes/cm. for the perfused preparation and 11 ± 3 for the minced lungs. Note that in the above cited paper by Morgan et al. (1965) minimum surface tensions in control dogs were given as 1 3 and 20 dynes/cm. In oxygen exposed dogs surface tensions were reported tobe 22 ± 3 for the perfused preparation and 26+5 for the minced lungs. Collier et al. (1965) exposed rabbits to one atmosphere of oxygen. They demonstrated from a minced lung extract that both by Pattle's bubble assay technique and by determining minimum surface tension on a Wilhelmy balance that the oxygen exposed rabbits showed statistically significant decreases in pulmonary surfactant. Effects of Oxygen Toxicity on Tissue Compliance

It was noted in the results (Table 9) that oxygen toxicity significantly reduced compliance due to tissue forces. Compliance wa3 determined from the slopes of the saline curves. The causes for this change are not immediately apparent. It may be due to the formation of perivascular edema, but this seems doubtful in view of the work of Cook, Mead et al. (1959) who showed that edema (pro­ duced by clamping of the aorta and the administration of dextran) actually increased tissue compliance above control values. In our own results it was shown that the process of perfusion, which pro­ duces edema, increased tissue compliance. Another possible cause is that of a change in alveolar wall such as the deposition of a hyaline membrane. Becker-Freyseng and Clamann (1939) reported that the lungs of cats and rabbits exposed to 600 mm Hg oxygen for seven days were edematous and lined with a debris-filled membrane. They reported that this membrane adhered to the vascular walls and ex­ tended into the bronchioles and appeared fibrinous in nature. In an electron microscope study of oxygen toxicity (Cedergren et al.,

1959), it was shown that one atmosphere of oxygen for three to six days in mice produced "an apparent patchy thickening of the alveolar wall due either to hypertrophy or fluid accumulation in the cells."

Lung Lecithin Values as an Index to Surface Activity

Quite a large number of investigators have studied the com­ position of surfactant, and many have related its lipid constituents 92 with its surface properties. Clements (1962) indicated that the

surface-active agent in the lungs is a lipoprotein, and that the fatty constituent is primarily lecithin. It was shown that dipal- mitoyl lecithin has the same surface characteristics as natural

surfactant. Clements pointed out that the same substance which was extracted from the lungs both gave characteristic low surface tension readings on the Wilhelmy balance and was primarily composed of lecithin (1962).

Brown (1962) showed that the surface-active substance extracted from the lungs which lowered surface tension contained choline which,

according to him, indicated that the surface-active material is an

alpha lecithin. Pattle and Thomas (1961) compared the extracted lung lining substance which produced stable bubbles with egg lecithin on

a spectrophotometer and found qualitatively identical curves for both

substances. They concluded: "It is clear that the lining film con­

sists in the main of a lecithin-protein complex." Klaus, Clements,

and Havel (1961) showed that surfactant bearing lung extracts and phospholipids (later shown to be primarily lecithin) produced identical

surface tension diagrams on a Wilhelmy balance. They also showed

that synthetic dipalmitoyl lecithin gave a similar diagram on the

Wilhelmy balance. The extraction process used in -this case to remove

the surfactant was the same as was used to obtain our results (3on-

durant, 1962).

Morgan et al. (1965) analyzed total lung lipids and "alveolar lipids," those removed by lavage, and reported, "We found marked 93

surface activity to be characteristic of highly saturated 'alveolar' lecithin and PX (unusual spot) fractions. Other lipid components

failed to show this activity.'1 Chu, Clements and Klaus et al. (1965) reported that in respiratory distress syndrome of the newborn there was a highly significant decrease in total lung lipids, choline-

containing phospholipids (mainly lecithin) and surface activity

(recorded as an increase in minimum surface tension). Fujiwara et al.

(1964) showed similar surface tension loops for both the crude ex­

tract and the phospholipid fraction of guinea pig lungs. Harlan

et al. (1966) reported that the total phospholipid content of the lung was significantly higher in situations with high surface activity.

Several species and varying degrees of atelectasis were compared.

Although the relative proportion of lecithin to phospholipid did not

change, lecithin was the major phospholipid, and hence it too was

highly correlated with changes in surface activity.

Methods of Extraction of Surfactant

A number of methods of extraction of surfactant have been used

with varying degrees of success. Common techniques are those of:

1) mincing and chopping, 2) homogenization, 3) lavage (foam fraction­

ation), and 4) perfusion-transudation. The results of surfactant

determinations by surface tension measurements or by lipid analysis

are probably highly dependent on the method of extraction employed.

Levine and Johnson (1964) compared three techniques of extraction and

pointed out several common inherent errors. Using both aerated and 94

atelactatic rabbit lungs, they compared the techniques of chopping, mincing, and pestle-homogenization. The tissue was extracted with

saline and filtered through gauze and the surface tension measured on a Wilhelmy balance and the stability index determined according

to Pattle’s method (1958) •

It was found that all three methods gave essentially similar results in aerated lung samples; all showed highly active extracts.

However in degassed lungs, the degassing being done in a vacuum

desiccator, only the pestle-homogenized lungs showed essentially

normal surface activity. Extracts from the chopping and mincing procedures showed higher minimum surface tensions and low stability indices. These results may be considered to imply that any condition

which results in total or partial atelectasis may obstruct surfactant

removal by the mincing and chopping processes.

Morgan et al. (1965) compared what he called "alveolar" lung lipids, those removed by saline lavage techniques, with total lung lipids, those removed by homogenization in chloroform-methanol. It

is unfortunate that a true comparison cannot be made of alveolar

and total lung lipids because two entirely different techniques were

employed. There is a comparison between lavage and homogenization

techniques added to the comparison between saline and chloroform

extractions. In any case, their results indicate an approximate

ten-fold difference between total alveolar lipids and whole lung lipids, and approximately an eight-fold difference between alveolar lecithin and total lung lecithin. Whole lung total lipid volumes in dog lungs were 1820 mg whereas alveolar lipid values were 228 mg

(Morgan et al.. 1965).

Klaus (1961) used Bondurant and Miller’s (1962) transudation technique and reported that the transudate contained 74 percent phospholipids and 5 percent nitrogen, and that it had good surface tension lowering characteristics. More recently, Giammona, ,'erner, and Bondurant (1965) compared mincing and foam fractionation (lavage) techniques in three species as to the effects of oxygen toxicity.

They reported that in cats and rabbits, both methods indicated that oxygen toxicity produced a lower surface activity. However, in rats it was reported that only the mincing technique showed a lower surface activity after oxygen toxicity, but that the homogenization technique indicated that the surface activity did not change. If these re­ sults are accepted, they would imply that when the mincing technique was used, especially in the case of oxygen toxicity, the results are questionable. This method has been used in several cases: Jamieson and Brenk (1964), on rats at five atmospheres of oxygen, Webb et al.

(1966) on rats and guinea pigs at 45 psi, Collier et al. (1965) on rabbits at one atmosphere, and Fujiwara et al. (1964) on guinea pigs at one atmosphere.

The technique used in this report was that of a transudation of saline across alveolar walls, developed by Bondurant and Miller

(1962). Briefly, the technique involves cannulation of the pulmonary artery, infusion of saline while the excised lung is being alter­ nately expanded and contracted by varying pressure around the lung, 96 and the application of a vacuum to the trachea from which the transu­ date is drawn. This transudate contains the dissolved or dispersed alveolar surface lining. It comes out of the trachea as a foam.

They reported the surface tensions at a minimum of 20 dynes/cm when sev­ eral extraction techniques were compared to be the following: homogen­ ization - 22 dynes/cm, saline washings - 21 dynes/cm, lung compressed in saline - 19 dynes/cm, tracheal effluent during distilled water perfusion - 21 dynes/cm, and tracheal effluent during saline perfu­ sion (the process used to obtain our results) - 11 dynes/cm. The lower minimum surface tension has been shown to correlate well with increased surface activity, or in this case, a more complete extraction of surfactant.

Effect of Edema on Air and Saline Pressure-Volume Curves

Since it is generally accepted that one of the effects of oxygen toxicity is pulmonary edema, and that edema fluid in the alveoli may alter pressure-volume curves, the question arises as to whether it is possible to separate alterations in surface forces from those due only to pulmonary edema. The pressure-volume characteristics of edematous lungs have been studied by several investigators. Hughes,

May and Widdicombe (1958) reported that induced pulmonary edema in perfused rabbit lungs to the extent of a 100 percent weight increase resulted in only a 30 percent reduction in total lung compliance.

The effects of 100 percent oxygen reported in our results showed that for only a 25 percent lung weight increase, there was a 50 percent 97 reduction in total lung compliance. It may be inferred from this comparison if rabbit and rat lung edema are considered essentially the same, that the edema which resulted from oxygen toxicity accounted for only a small part of the reduction in compliance, less than 8 percent.

The other 42 percent, not accounted for by the presence of edema fluid, was presumably primarily due to Increased surface tension.

Hemingway and Williams (1952) studied the effects of oxygen toxicity on the production of pulmonary edema in guinea pigs. After

48 hours of exposure to 100 percent oxygen they found a doubling in lung weight (our results indicated a 25 percent increase after 60 hours in rats), and a doubling of soluble protein nitrogen. The amount of insoluble protein nitrogen remained essentially the same as did hemoglobin levels. It was concluded that the weight increase was due to the presence of edema fluid, and not due to capillary proliferation or filling with blood. The reason for the large difference in the amount of edema fluid in rats and guinea pigs resulting from oxygen toxicity is not known. It should be noted that Fujiwara et al. (1984) in a study of the effects of oxygen toxicity in guinea pigs, reported similar changes in soluble protein nitrogen levels but found no significant changes in either lung lipids or in the minimum surface tension of minced lung extracts.

Cook, Mead et al. (1959) approached the problem of the separ­ ation of the effects of surface forces and edema fluid on lung compliance more directly. They produced pulmonary edema In dogs by partial aortic obstruction and intravenous infusion. The immediate 98 effect was slight, but after time was allowed for edema to develop, a 78 percent reduction in compliance developed (our results indicated a 50 percent reduction in compliance after oxygen toxicity). How­ ever, they calculated on a 6 percent decrease in compliance if allowance was made for edema fluid and trapped gas. Similar cal­ culations made on our results indicated a 33 percent decrease in compliance. Cook, Mead et al. (1959) concluded that "these findings suggested that surface phenomena were responsible for the mechanical behavior of edematous lungs rather than vascular congestion, per se, or intrinsic tissue changes." They reported a significant difference in the shape of the air pressure-volume curve after induced edema.

The first third of their inflation curves were clearly much flatter

(less compliant) in the case of the edematous lung, but the middle third of the curve was actually steeper than the corresponding air curve, thus overcoming the initial flatness and resulting in a total compliance only slightly different from that of the air curve. In our oxygen toxic lungs the situation was quite the reverse. The first third of the inflation curve was essentially identical with the air curve, but the middle and last third of the curve was much flatter than the control curve. Total compliance was considerably less

(33 percent compared with their 6 percent) after both curves were adjusted for edema fluid and residual air.

Thus again it appears that the production of pulmonary edema cannot by itself account for the altered pressure-volume curve and decreased compliance. Furthermore, Pattle (1958) and Brown (1957) 99 have given indirect evidence that surfactant is essentially the same in edematous and normal lungs. Pattle concluded from the stability of edema foam and of bubbles expressed from normal lungs that surface tension was extremely low in both instances. Brown studied pulmonary edema fluid and extracts of normal lungs and found their surface behavior to be essentially the same.

Oxygen Toxicity and Atelectasis - Lung Residual Volumes

It was found that there was no significant difference in residual volume in control and oxygen poisoned lungs (Table 10).

It has been reported extensively (Bean, 19^5) that oxygen toxicity causes pulmonary atelectasis. The reason for this apparent dis­ crepancy between our results and those of most other investigators is not entirely clear. The difference may lie in the techniques of measurement or may be due to the fact that in most work reported on tjy Bean, the animals died in oxygen. Most, but not all, reports on atelectasis due to oxygen toxicity are based on the blood-filled, liver-like appearance of the lungs. One study was based on the lung volume at 5 cm* water pressure after a pressure-volume curve was run (Clements et al.. 1961). Our results were based on the buoyancy of the lungs suspended in water. Buoyancy was quantitated by measur­ ing the uplift of the lungs (see Procedures). It was found that control and oxygen poisoned lungs were consistently the same. If oxygen toxicity is severe to the point of death or near death, the lung frequently contains no residual air as was found by us and by others (Bean, 19^5). 100

Static and Dynamic Compliance Measurements

The term dynamic compliance has been applied to the compliance value determined at the two points in a normal respiratory cycle when air flow is instantaneously zero, that is, at the beginning of inspiration and at the beginning of expiration (Mead, 1961)* Other terms in use for this same value are equivalent compliance, effective compliance, functional compliance, and just compliance. Five or more studies have shown that compliance determined by the method described above gives the same values from static conditions to frequencies as high as 60 to 90 breaths (or cycles in excised lungs) per minute

(Mead, 1961). Otis, Mead, Radford et al. (1956) showed in humans that when the compliance of the lungs was measured as the ratio of volume change to the difference in pressure at the instants of maximum and minimum volume, rates from zero (static compliance) to 120 cycles per minute did not alter the compliance. In our study, the cycle frequency was kept very low, only 3 cycles per minute. Thus it may be assumed that our measurements represent the true compliance as would be found under static conditions.

Method of Lipid Analysis

Amenta's method (1964) of lipid analysis was used for the quantitation of lecithin, after lecithin had been separated by thin layer chromatography. Egg lecithin was used as a standard, shown by

Pattle and Thomas (1961) to give a qualitatively identical spectrum on the infrared spectrophotometer as did pulmonary surfactant. 101

Freeman and West (1966) recently demonstrated that .Amenta's method was very successful in their hands using lecithin and other lipids separated by thin layer chromatography. However, they reported that a decrease in saturation affected their results. It is known (Morgan et al.. 1965) that oxygen poisoning decreases the degree of saturation of surfactant lipids (lecithin) by as much as 35 percent. Freeman and

West reported that a 100 percent decrease in the degree of saturation decreased the apparent outcome of the lipid quantitation by 7 per­ cent. Thus it can be assumed that a 35 percent reduction in saturation would decrease the apparent outcome of the lipid quantitation by less than 7 percent, approximately 2 to 3 percent. In our results, oxygen poisoning resulted in greater than a 100 percent decrease in lecithin.

The change in lecithin was from 623 micrograms to 293 micrograms. By the above reasoning, 2 to 3 percent of this decrease could logically be assigned to the change in saturation.

Saline Inflation and Deflation Curves

Deflation saline curves have been used consistently to gather data on the tissue properties of lungs. In our study both inflation and deflation curves were analyzed. Although the results are dif­ ferent between the two curves, the same conclusions are reached.

Comparing control and oxygen rats, inflation curves (Table 9) show a compliance of 1.80 ml/cm water in controls and 1.3 0 in oxygen rats.

Using the deflation curves, controls show values of 0.60 ml/cm water and oxygen exposed rats show values of 0.24 ml/cm water. In either 102

case the difference after oxygen exposure is significant. The reason

for the difference between inflation and deflation curves is not known.

When surface compliance values are calculated using either curve, the results are similar. There is a significant decrease in surface

con^liance due to oxygen poisoning in either case (Table 9). In the

case of air curves, the points of minimum and maximum inflation are used as the basis for the compliance value, and these points are

identical for the inflation and deflation curves. Thus, this problem only exists when saline curves are considered. SUMMARY

The effect of oxygen toxicity on rat lungs was investigated by:

1. Exposing approximately 100 rats of average body weight of

215 grams to 1 atmosphere of 100;' oxygen for 60-66 hours in

a closed recycling chamber.

2. Seoaration of tissue effects from effects of surface forces

by pressure-voliune diagrams made by inflating lungs, with air

and saline, both before and after extraction of surfactant.

Compliance values were calculated from the pressure-volume

curves.

3. Examining changes in surfactant by extracting the surface

lining via perfusion through the pulmonary circulation, col­

lecting the tracheal transudate thus formed, and quantitating

the lecithin in the transudate by thin layer chromatography.

The results indicated that:

1. Perfusion of control rat lungs removed surfactant and reduced total lung compliance and the component of compliance due to surface forces. Total compliance was reduced from 0.15 ml/cm. water to 0.09 and the surface component of compliance from 0.21 ml/cm. water to 0.11.

The component of compliance due to tissue forces did not show a signif­ icant change. Lecithin extracted from the perfusate (trandudate), taken as an index of surfactant amounted to 623 micrograms per rat.

2. Exposure of rats to one atmosphere of oxygen reduced total 103 104 compliance, both components of compliance, and lung lecithin signifi­ cantly. Total compliance was reduced from 0.07 ml/cm. water to 0.04, the compliance due to tisstie forces was reduced from 1.80 ml/cm. water to 1.30. Lung lecithin was reduced by and averaged 293 micrograms per rat. A significant correlation of 0.82 was found between the compon­ ent of compliance due to surface forces and lung lecithin.

3. Removal of surfactant by perfusion did not effect oxygen rat lungs as much as controls. In oxygen rats total compliance decreased from 0.07 to 0.04 and the component of compliance due to surface forces did not significantly change. It is generally concluded that oxygen toxicity decreased lung compliance both by decreasing surfactant and by increasing tissue rigidity. BIBLIOGRAPHY

Amenta, J. S., "A rapid chemical method for quantification of lipids separated by thin-layer chromatography," J. Lipid Res., 5: 270- 272, 1964.

Arias-Stella, Javier and Hever Kruger. "Pathology of High Altitude Pulmonary Edema," Arch. Path., 7 6 : 147-157* 1963.

Avery, K.E. "The Alveolar Lining Layer, A Review of Studies on its Role in Pulmonary Mechanics and the Pathogenesis of Atelectasis," Pediatrics, 30: 324-330, 1962.

Bayliss, L. E. and G. W. Robertson. "Viscoelastic Properties of the Lungs," Quart. J. Expl. Physiol., 29: 27-47. 1939*

Bean, J. W. "Effects of Oxygen at Increased Pressure," Physiol. Rev., 25: 1-147, 1945.

Becker-Freyseng, II. and H. G. Clamann. "Zur frage der Sauerstof- fuergiftung," Klin. Wschr., 18: 1382-1385, 1939«

Bert, P. "La Pression Barometrique," Paris: G. Masson, 1878.

Bert, P. "Barometric Pressure — Researches in Experimental Physio­ logy," translated from French by K. A. Hitchcock and F. A. Hitchcock, Columbus: College Book Co., 1943-

Bondurant, 3. and 0. A. Miller. "A method for producing surface active extracts of mammalian lungs," J. Aopl. Physiol., 17: 167-168, 1962.

Bondurant, 3. and C. Smith, "Effect of Oxygen Intoxication on the Surface Characteristics of Lung Extracts," The Physiologist, 5-3: 111, 1962.

Bragdon, J. H. "Colorimetric Determination of Blood Lipids," J. Biol. Chem., 190: 513-517, 1951.

Brotm, E. 5. "Lung Area from Surface Tension Effects," Proc. Soc. Exper. Biol. Med., 95: 168-172, 1957.

Brown, E. 5. "Assay of Surface Active Material from Emphysematous Lungs," Med. Thorac., 22: 70-76, 1965

105 106

Brown, E. 3. "Chemical Identification of a Pulmonary Surface Active Agent," Fed. Proc., 21: 329, 1962.

Butler, J., H. C. bhite and ¥. M. Arnott, "Pulmonary Compliance in Normal Subjects," Clin. Sci., 16: 709-729. 1957.

Caldwell, P.R.B., S. T. Giammona, Vf. L. Lee, Jr. and S. Bondurant. "Effect of Oxygen Breathing at One Atmosphere on Pulmonary Surfactant in 7'ogs," Clin. Res., 11: 301, 1963.

Cedergren, B.,L. Gyllensten and J. Uersall. "Pulmonary Damage Caused by Oxygen Poisoning," Acta Pediat., h8: b77-h9h, 1959*

Cherniack, R. K. "Physical Properties of the Lung in Chronic Obstruc­ tive Pulmonary Emphysema," J. Clin. Invest., 35: 39^-^Oh, 1956.

Cherniack, R. M. and A. Hodson. "Compliance of the chest wall in chronic bronchitis and emphysema," J. Appl. Physiol., IS: 707- 711. 1963.

Chu, J., J. A. Clements, E. Cotton, M. K. Klaus, A. Y. Sweet, F. A. Thomas and 7/. K. Tooley. "The Pulmonary Hypoperfusion Syn­ drome," Pediatrics, 35: 733-7^2, 1965.

Clements, J. A. "Dependence of Pressure-Volume Characteristics of Lunns on Intrinsic Surface-active Material," Am. J. Physiol., 187: 592, 1956.

Clements, J. A. "Surface Tension of Lung Extracts," Proc. Soc. Exper. Biol. Ked. , 95= 170-172, 1957-

Clements, J.A. "Pulmonary Edema and Permeability of Alveolar Fem- branes," Arch. Envir. Health, 2: 280-283, 1961.

Clements, J. A. "Surface Tension in the Lungs," Scien. Amer., Ib2: 2-9, 1962

Clements, J. A., R. F. Rusted, R. P. Johnson and I. Gribetz. "Pul­ monary Surface Tension and Alveolar Stability," J. Appl. Physiol., 16: Wl-fy50, I96I.

Clements, J. A. and H. J. Trahan. "Effects of Temperature on Pres- sure-Volume Characteristics of the Lungs," Fed. Proc., 22: 281, 1963.

Collier, C. R., J. D. Hackney and D. E. Rounds, "Alterations of Sur­ factant in Cxygen Poisoning," Pis. Chest, h-8: 233-238, 1965* 107

Cook, C. D., J, Mead, G, L, Schreiner, N. R. Frank and J, M, Craig, "Pulmonary mechanics during induced pulmonary edema in anes­ thetized dogs," J. Appl. Physiol., 14: 177-186, 1959*

Cropp, G. J. A. "Effect of high intra-alveolar O2 tensions on pul­ monary circulation in perfused lungs of dogs," Amer. J, Physiol.. 208: 130-138. 1965-

Drinker, C. K. "Pulmonary Oedema and Inflammation," Cambridge: Harvard University Press, 1950.

Faridy, E. E., S. Permutt and R. L. Riley, "Effect of ventilation on surface forces in excised dogs1 lungs," J. Appl. Physiol., 21: 1453-1462, 1966.

Fedde, M. R. and R. E. Burger. "Death and Pulmonary Alterations Following Bilateral Cervical Vagotomy in the Fowl." Poultry Sci.. XLII: 1236-1246, 1963.

Finley, T. N., W. H. Tooley, E. W. Swenson, R. E. Gardner and J. A. Clements. "Pulmonary Surface Tension in Experimental Atel­ ectasis," Amer. Rev. Resp. Pis., 89: 372-378, 1964.

Folch, J., M. Lees and G. H. S. Stanley. "A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues," J.3.C.. 226: 497-509, 1957.

Frank, N. R. "Influence of acute pulmonary vascular congestion on recoiling force of excised cats' lung," J. Appl. Physiol.. 14: 905-908, 1959.

Frank, N. R. "A Comparison of static volume-pressure relations of excised pulmonary lobes of dogs," J. Appl. Physiol., 18: 274- 278, 1963.

Frank, N. R., E. P. Radford, Jr. and J. L. Whittenberger. "Static volume-pressure interrelations of the lungs and pulmonary blood vessels in excised cats' lungs," J. Aopl. Physiol.. 14: 167-173, 1959.

Freeman, C. P. and D. West. "Complete separation of lipid classes on a single thin-layer plate," J. Lipid Res.. 7: 324-326, 1966.

Fujiwara, T., F. H. Adams and A. Scudder. "Fetal Lamb Amniotic Fluid: Relationship of Lipid Composition to Surface Tension," J. Ped.. 65: 824w830, 1964.

Fujiwara, T., F. H. Adams and K. Seto. "Lipids and surface tensions of extracts of normal and oxygen-treated guinea pig lungs," J. Ped.. 65: 45-52, 1964. 108

Giammona, S. T. and S. Bondurant. ’’Comparison of duNuoy Tensiometer and Wilhelmy Balance for Measuring Surface Tension of Pulmonary Surfactant," J. Lab. Clin. Med.," 65 : 329-333, 1965.

Giammona, S. T., R. Frayser, P. R. Caldwell and S. Bondurant. "Effect of Oxygen Intoxication on Pulmonary Surfactant in Dogs, Rabbits, and Rats." J. Ped., 65: 118-119, 1964.

Giammona, S. T., D. Kerner, and S. Bondurant. "Effect of oxygen breathing at atmospheric pressure on pulmonary surfactant," J. Appl. Physiol., 20: 755-858, 1965.

Giammona, 3. T., I. Nandelbaum, J. Foy and S. Bondurant. "Effects of Pulmonary Artery ligation on Pulmonary Surfactant and Pressure- Volume Characteristics of Dog Lung," Circul. Res., XVIII: 683- 691, 1966.

Gitlin, D. and J. M. Craig. "The Nature of the Hyaline Membrane in Asphyxia of the Newborn," Pediatrics, 17: 64-71, 1956.

Gruenwald, Peter. "Normal and Abnormal Expansion of the Lungs of New­ born Infants Cbtained at Autopsy, Expansion of Lungs by Liquid Media," Anat. Rec., 139: 4?lJ'79,~1961.

Gruenwald, Peter. "Pulmonary urfa.ee Forces as Affected by Tempera­ ture," Arch. Path., 77: 568-574, 1964.

Hackney, J. D., D. E. Rounds and A. If. Schoen. "Observations of a lipid Lining in Mammalian Lung," Fed. Proc., 22: 339, 1963.

Harlan, V. R., Jr., J. H. Margraf and S. I. Said. "Pulmonary lipid composition of species with and without surfactant," Amer. J. Physiol., 211: 855-862, I966.

Hemingway, Allan. "A Method of Chemical Analysis of Guinea Pig Lung for the Factors Involved in Pulmonary Edema," J . Lab. Clin. Med., 35: 817-822, 1950.

Hemingway, Allan. "Pulmonary Edema in Guinea Pigs During Severe Kyposia," J. Appl. Physiol., 4: 868-872, 1952

Hemingway, A. and W. L. Williams. "Pulmonary Edema in Oxygen Poison- ing," Soc. Exoer. Biol. Med., 80: 331-334, 1952.

Hovatt, V. E., K. E. Avery, P. V. Humphreys, I.C.5. Normand, L. Reid and L. B. Strang. "Factors Affecting Pulmonary Surface Proper­ ties in the Foetal Lamb," Clin. Sci., 29: 239-248, 1965* 109

Hughes, R., A. J. May and J. G. Widdicombe. "The Effect of Pulmonary Congestion and Edema on Lung Comoliance," J. Physiol., 142: 306- 313^ 1958.

Hulpieu, H. R. and V. V. Cole. "The Effect of Humidity and Temperature on Oxygen Toxicity," J. Lab. Clin. Med., 29: 1134-1138, 1961.

Jamieson, D. and II. A. S. van den Brenk. "Pulmonary Damage due to High Pressure Oxygen Breathing in Rats," Aust. J. Exp. Biol. Med. Sci., kz-. 483^ 90. 1964

Karsner, H. T. and J. E. Ash. "A Further Study of the Pathological Effects of Atmospheres Rich in Oxygen," J. Lab. Clin. Med., 2: 254-255, 1916.

Kennedy, J. H. "Hyperbaric Oxygenation and Pulmonary Damage, The Effects of Exposure at Two Atmospheres upon Surface Activity of Lung Extracts in the Rat," Ked. Thorac., 23: 27-35, 1966.

King, T. K. C. "Mechanical properties of the lungs in the rat," J. Appl. Physiol., 21: 259-264, 1°66.

Klaus, K. P., J. A. Clements and R. J. Havel. "Composition of Surface- Active Material Isolated from Beef Lung," Proc. Hat. Acad. Sci., 4?: 1858-1859, 1961.

Lance, J. S. and H. Latta. "Hypoxia Atelectasis and Pulmonary Edema," Arch. Path., 75: 373-377, 1963.

Levine, B. E. and R. P. Johnson. "Surface activity of saline extracts from inflated and degassed normal lungs," J. AppI. Physiol., 19: 333-335. 1964.

Levine, B. E. and R. P. Johnson. "Effects of atelectasis on pulmonary surfactant and quasi-static mechanics," J. Appl. Physiol., 20: 859-864, 1965.

Mcllroy, H. B. "Physical Properties of ilormal Lungs Removed after Death," Thorax, 7: 285-290. 1952.

Mead, J. "Mechanical Properties of Lungs," Physiol. Rev., 41: 2.81- 330, I96I.

Mead, J., J. L. Whittenberger and E. P. Radford, Jr. "Surface Ten­ sion as a Factor in Pulmonary Volume-Pressure Hysteresis," J. Appl. Physiol., 10: 191-196, 1957.

Miller, D. A. and S. Bondurant. "Surface characteristics of verte­ brate lung extracts," J. Appl. Physiol., 16: 1075-1977, 1961. 110

Morgan, T. E., T. N. Finley and H. Fialkow. "Comparison of the Com­ position and Surface Activity of 'Alveolar1 and Whole Lung Lipids in the Dog," Biochlra. Biophys. Acta, 106: 403-413. 1965.

Morgan, T. E., T. N. Finley, G. L. Huber and H. Fialkow. "Alterations in Pulmonary Surface Active Lipids during Exposure to Increased Oxygen Tension," J. Clin. Invest.. 44: 1737-1744, 1965.

Neergaard, K. V. "Neve Auffassungen uber einen Grundbergriff der Aternmechanik die Ratraktionskraft der Lunge, Abhangig von der Oberflachenspannung in dem Alveolen," Zeitsch. dur die Ges. Exper. Med.. 66 : 373-394, 1929.

Nouy, P. L. du. "Surface Equilibria of Colloids," New York: Rein­ hold, 1926.

Otis, A. B., B. Colin, C. B. McKerrow, R. A. Bartlett, J. Mead, M. B. Mcllroy, N. J. Selverstone and E. P. Radford. "Mechanical Fac­ tors in Distribution of Pulmonary Ventilation," J. Appl. Physiol. 8: 427-443, 1956.

Otis, A. 3., C. B. McKerrow, R. A. Bartlett, J. Mead, M. B. Mcllroy, N. J. Selverstone and E. P. Radford, Jr. "Mechanical Factors in Distribution of Pulmonary Ventilation," J. Appl. Physiol.. 8: 427-432, 1956.

Pattle, R. E. "Properties, Function and Origin of the Alveolar Lining Layer," Nature. 175: 1125-1126, 1955.

Pattle, R. E. "A Test of Silicone Anti-foam Treatment of Lung Edema in Rabbits," J. Path. Bacteriol.. 72: 203-209, 1956.

Pattle, R. E. "Properties, function, and origin of the alveolar lining layer," Proc. Roy. Soc. B. 148: 217-240, 1958.

Pattle, R. E. "The Lining Layer of the Lung Alveoli," Brit. Med. Bull.. 19: 41-44, 1963.

Pattle, R. E. "Surface Lining of Lung Alveoli," Physiol. Rev.. 45: 48-79. 1965.

Pattle, R. E. and F. Burgress. "The Lung Lining Film in Some Path­ ological Conditions," J. Path. Bacteriol., 82: 315-331. 1961.

Pattle, R. E. and D. A. W. Hopkinson. "Lung Lining in Bird, Reptile and Amphibian," Nature. 200: 894, 1963. 111

Pattle, R. E. and L. C. Thomas. "Lipoprotein Comparison of the Film lining the Limp," Nature, 189: 844, 1961.

Radford, 5. P., Jr. "Method for Estimating Respiratory Surface Area of Mammalian Lungs from their Physical Characteristics," Proc. Soc. Exper. Biol. Med., 87: 53-61, 195^•

Radford, E. P., Jr. "Recent Studies of Mechanical Properties of Mammalian Lungs," in: Tissue Elasticity, J. W. Remington (ed.), Washington, D. C.: American Physiological Society, 1957*

Radford, E. P., Jr. "Mechanical Stability of the Lung," Arch. Env. Health, 6: 128-133, 1963.

Rhoades, R. A. "Metabolic Changes in Animals Exposed to a Helium- Cxygen Environment," A Thesis, The Chio State Lniversity, Columbus, 1966.

Robillard, E., Y. Alarie, P. Paganuzzi and L. Dautrebande. "New Studies on Aerosols, XXI. 'Pressure-Yolume' Curves Obtained on Isolated Atelectatic Rats’ lung after Short Inhalations of Various Insoluble Submicronic and Submicroscopic Particles," Arch. Int. Pharmacodyn., 1 4 7: 220 -2 2 9 , 1964.

Rosenberg, E. ,Y.Alarie and T?. Robillard. "Effect of Rust and Aero­ sol Inhalation on Surface and Tissue Elasticity of Rat Lungs," Canad. J. Biochem. Physiol., 40: 1359-1365, 1962.

Roth, E. M. "Space Cabin Atmosphere, Part I. Oxygen Toxicity." NASA, 3P-47.

Said, 5. I., R. I.Davis, M. Davis, C. K. Banerjee and K. El C-ohary, "Pulmonary Surface Tension and Morphologic Changes in Acute Pul­ monary Edema," Fed. Proc., 2 2 : 339, I963.

Scarpelli, E. K . , K. H. Babbay and J. A. Kochen. "Lung Surfactants, Counterions, and Hysteresis," Science, 148: 1607-1609, 1965.

Schaefer, X. E., M. E. Avery and K. Bensch. "Time Course of Changes in Surface Tension and Morphology of Alveolar Epithelial Cells in CCM-induced Hyaline Membrane Disease," J. Clin. Invest., 43: 2080-2093, 1964.

Schoedel, W. von. "Einflusse von Beatmung und Zigarettenrauch auf das statische Druck-Volumen-Diagranmisolierter Rattenlungen," Pflurers Archiv., 284: I76-I83, 1965• 112

Setnikar, I. "Keccanica Respiratoria Fstratto dal," Vol. 3. Degli Aggiornamenti di Fisiologia, Dtt. Luigi Kacri, ed. Pb, Firenze, 1955-

Sharp, J. T., G. T. Griffith, I. L. Bunnell and D. G. Greene. "Venti­ latory Mechanics in Pulmonary Edema in Kan," J. Clin. Invest., 37: 111-117, 1958.

Smith, L. "The Pathological Effects Due to Increase of Cxygen Ten­ sion in the Air Breathed," J. Physiol., 2b: 19-351 1899*

Snedecor, George W., Statistical Methods, Ames, Iowa: The Iowa State University Press, 1956.

Sutnick, A. I. and L. A. Soloff. "Surface Tension Reducing Activity in the Normal and Atelectatic Human Lung," Amer. J. Ned., 35: 31- 36, 1963.

Swann, H. E., Jr. "Occurrence of Pulmonary Edema in Sudden Asphyxial Deaths," AKA Arch. Path., 69: 557-570, i960.

Thannhauser, 3. J., J. Benotti and N, F. Boncoddo. "Isolation and Properties of Hvdrolecithin (Dipalmityl Lecithin) from Lungs; Its Occurrence in the Snhingomyelin Fraction of Animal Tissues," J. Biol. Chem., 166: 669-715. 19'kS.

Tierney, D. F. "Pulmonary Surfactant in Health and Disease," J . Appl. Physiol., b?: 247-253-

Tierney, D. F. and R. P. Johnson. "Altered surface tension of lung extracts and lung mechanics," J. Appl. Physiol., 20: 1253- 1259, 1965.

Tooley, W . , R. Gardner, H. Thung and T. Finley. "Factors Affecting the Surface Tension of Lung Extracts," Fed. Proc., 20: b28, 1961.

Tooley, V. H., C. Piel and J. A. Clements. "Alveolar Epithelial Cell Mitochondria as Source of the Surface-Active Lung Lining," Science, 137: 750-751. 1962.

Van de Woestijne, K. P. and J. P. Naedts. "Influence of Forced In­ flations on the Pressure-Volume Curve of the Lungs and Thorax in the Dog." Inter. Physiol. Biochim., 73: 593-609, 1965.

Webb, W. R., J. U. Lanius, Z. Aslami and R. C. Reynolds. "The Effect of Hyperbaric-Cxygen Tensions on Pulmonary Surfactant in Guinea Pigs and Rats," J . Amer. lied.Assn., 195: 279-280, 1966. 113

Weir, F. W., D. W. Bath, P. Yevich and F. W. Oberst. "A Study of the Effects of Continuous Inhalation of High Concentrations of Oxygen at Ambient Pressure and Temperature," ASD-TR-6l-66*+, 1961.

Weir, F. W., D. W. Bath, P. Yevich and F. W. Oberst, "Study of Effects of Continuous Inhalation of High Concentrations of Oxygen at Ambient Pressure and Temperature," Aerospace Med., 36 : 117- 12 0 , 1965.

Weiss, H. S., R. A. Wright and E. P. Hiatt. "Embryo Development and Chick Growth in a Helium Oxygen Atmosphere," Aerospace Med., 36: 201 -206, 1965.

Wright, R. A., H. S. Weiss, E. P. Hiatt and J. S. Rustagi. "Risk of mortality in interrupted exposure to 100$ 0 3 : role of air vs. lowered PO2 ," Amer. J. Physiol.. 21: 1015-1020, 1966.