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Surface properties of fetal lamb tracheal fluid
Surface properties of fetal lamb tracheal fluid G6RAN ENH6RNING, M.D. F 0 R R E S T H . AD AM S , M .D . Los Angelts, California
F r R s T breaths of the newborn infant are the most difficult. 1 Intraesophageal pressure recordings indicate that they offer much greater resistance than subsequent inspirations.2 Prim· to the first breath the trachea and the finer branches of the airvvays arc filled with liquid. When the intrathoracic pressure is lowered in an attempt to aerate the lungs, this liquid has to move ahead of the air and its viscosity and surface tension is an obstacle to these first breaths. Few studies have been focused on the resistance that surface tension of the airway liquid offers to the first breath. 3 - 5 More interest has been given to its importance once respiration has been established. 6 ' 12 The alveolus will close with exponential speed if the surface tension of its lining layer remains at a fixed value. There is good evidence, however, that surface tension decreases as the alveolus becomes smaller and the tendency of the alveolus to close is lessened. 8 - 13 There is also good evidence that this ability to stabilize the alveoli by a substantial decrease of surface tension with decreased alveolar size is absent or inadequate in newborns dying of the respiratory distress syndrome.13 One of the major problems of studying this aspect of surface tension lies in the difficulty of obtaining a sample of the liquid in the air-liquid interface of the alveoli. Usually
the whole lung is cut into small pieces and stirred with physiological saline solution. This gives an extract of which 50 ml. is required for a study with the modified Wilhelmy balance. 9 ..A1dams and co-\vorkers 14 ' 1 u have desciibed a flow of liquid from the trachea in fetal lambs. At the time of the first breath this liquid must be pulled into the alveoli where it participates in forming a noncellular layer. If it has the ability of lowering surface tension with decreasing alveolar size, it will help to stabilize the alveoli. In this study, the surface properties of tracheal fluid collected from lambs in utero were evaluated. The surface tension, as it may affect resistance to the first breath was determined. The apparent changes in surface tension, which may occur during respiration, were recorded with a method which simulates alveolar ventilation. The alveolar stabilizing property of the surface layer was evaluated from these recordings and was also studied with a more direct approach.
THE
Materials and methods Collection of tracheal fluid. Six lambs from 6 ewes were used for this study. Five of the ewes were anesthetized with pentobarbital sodium injected into the jugular vein which had been dissected free under local anesthesia. An initial dose of from 0.5 to 0.65 Gm. was administered and supplemental doses of 0.5 Gm. were given as needed. A tracheotomy was done so that 100 per cent oxygen could be supplied to the ewe. A Harvard respirator was used. Its stroke volume was set at 350 mi. and its frequency at 24 to 28 strokes per minute. The blood pressure
From the Department of Pediatrics, School of Medicine, University of California, Los Angeles. Supported in Part by grants from United Cerebral Palsy Research and Educational Foundation (Dwight D. Eisenhower Fund) and the United States Public Health Service.
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was monitored during the operation and was consistently found to be within normal values. Ten minutes after the respirator was started, a blood sample was taken from the carotid artery for pH and pC0 2 determinations by the micromethod of Astrup and associates.1'; The values were within the normal range* in 5 sheep, but in one ewe, the one with Iamb No. 1 of this study, a decreasing pH and an increasing pC0 2 indicated inadequate ventilation. The cause was found and eliminated but in this ewe there had thus been a short period of severe asphyxia in the initial part of the experiment. During the operation the ewe was lying on her right side. The abdominal wall was incised in the midline and the pregnant uterine horn marsupialized to the edge of the abdominal wound. The uterus was then opened and the head of the Iamb was delivered. The trachea was exposed, and when it was opened just below the larynx, clear fluid poured out under pressure. A special container was used for its collection. It consisted of a rubber glove finger threaded over and tightly secured to the ends of two pieces of vinyl tubing. One tubing was short and wide (length, 10 em.; outer diameter, 0.5 em.), and when it had been introduced into the trachea and tightly secured, it gave free outlet to the rubber container. This container could be emptied intermittently through the other tubing which was long and narrow (length, 50 em.; outer diameter, 0.2 em.). After the tracheotomy and the introduction of the wide tube into the trachea, the head of the lamb as well as the rubber container were returned into the uterus which was closed, as was the abdominal wall. However, the narrow tubing formed an outlet through the uterine and abdominal incisions (Fig. I). In one ewe, the mother of lamb No. 5 of this study, the operative procedure was slightly different. In this animal a total of 1.3 Gm. of pentobarbital sodium was injected into one of the dilated abdominal veins. Without doing· a tracheotomy or using *Normal values in the unanesthetized, standing, term ewe for our laboratory range as follows: pH = 7.41 to 7.48; pCO, = 29 to 34 mm. Hg.
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the respirator, the operation was performed as described above. Howe\·er, in this case a hind leg was also delivered so that a catheter could be introduced into the femoral artcrv. This catheter together with the tubing from the collecting rubber container led through the abdominal wall. When the latter had been closed the ewe was allowed to wake up. Within an hour she was standing and che11ing her cud. Samples of tracheal fluid and blood for pH and pC0 2 determinations were thus obtained from the fetus while the f'WP was unanesthetized. Determination of surface tension as it affects resistance to the first breath. A modification of the maximum bubble pressure method was used. The technique has previously been described in a study on amniotic fluid 1 ; and therefore only its principal i'i outlined. Two small chambers, one containing water, the other thP sample, communicated with open air via fine bore n'ttical glass capillaries which had identical dimensions at the tip, inside the chamber. The capillaries contained liquid but as the chambers were slowly evacuated one at a time. a meniscus mo\'ed towards the capillary tip. The chambt'r was connected to a pressun~ transducer which recorded t}w negatin~ pressure which came to a peak ( ~ P) and became less negative as air mC)\'Cd to the capillary tip and a bubble was suddenly formed. Surface tension ( Ys I of the sample was calculated from the pressure dett'rmination since it is proportional to the surface tension of water ( Yw) as the peak pressure in the sample chamber (:l P,l is proportional to that in the water chamber ( ~ Pw) ; i.e.: y s
t. P,
y w
t. 1\,.
from which Ys was calculated since Yw is 72 dynes per centimeter at 20° C. Determination of surface properties of a bubble in tracheal fluid. A model of an alveolus and its airway was constructed which simulated the respiratory changes in akeolar size such that the surface properties could be evaluated. As shown in Fig. 2, this consisted of a chamber for the sample fluid which
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Fig. 1. Method for collec ting fetal lamb tracheal fluid in utero.
communicated with open air via a vertical glass capillary, the "airway." The chamber also communicated with a micrometer syringe, a pressure transducer and a pulsator unit. It was originally filled with the sample fluid, but when some of this was withdrawn with the micrometer syringe, air was sucked into the capillary and a bubble, the "alveolus," was formed in the chamber. At a frequency of 17 cycles per minute the pulsator moved 0.21 mm. 3 of liquid in and out of the chamber. This gave a cyclic change in bubble size. Each time the bubble was of minimal size, a mark was automatically made on the pressure record, and it was known that the maximal bubble size was exactly half way between two moments of minimal size, thus, half way between two marks. The pressure chamber was mounted on the objective table of a microscope and was moved in relation to the latter so that the op tical axis of th e microscope went through the center of the bubble. In this way the diameter of the bubble could be measured with a scale in the eye-piece of the microscope. A detailed description of the method is published elsewhere. 18 The chamber was fill ed with physiological saline solution and calibrated at -2 and -4 em. of water. By withdrawing with the
micrometer syringe a bubble was formed at the tip of the capillary. As the pulsator unit was started, the micrometer syringe was adjusted so that the maximal diameter of the bubble was 1,000 f.L· The minimal diameter was then ap proximately 840 p.. When the chamber contained physiological saline solution, this cyclic change in bubble size gave a characteristic symmetrical pressure tracingdemonstrating that the sample chamber had been adequately cleaned. The chamber was then calibrated at -1 and -3 em. of water and the physiological saline solution replaced with a sample of tracheal fluid . Again a bubble with a diameter of 1,000 p. was made at the capillary tip and the bubble was kept at this size while the pressure was recorded for a few minutes. The bubble was then extruded and replaced with a new one. The pulsations were started and continued for 30 minutes while the pressure was continuously recorded. Adjustments with the micrometer syringe secured a maximal bubble diameter of 1,000 f.t. When the pulsator unit was stopped, the bubble diameter was kept stable at 1,000 p. for approximately 2 minutes. The calibration was controlled at the end of each determination. With knowledge of the pressure gradient (~ P ) required to keep the bubble expanded,
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.I Hill' h . I ~~b Uhst. & Gyncc
.1
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ACriiAL PRESSIJREIIECOROOF WArEIIBIJBBLE
Fig. 2. Principle (a) and arrangement (b) for studying surface properties of a bubble in a liquid. The bubble communicates with atmosphere via a glass capillary and its size is determined with a scale in the eyepiece of the microscope. The bubble becomes large r and smaller as th e pulsator unit moves a small amount of liquid in and out of the chamber, simulating relative changes in alveolar size during respiration. Thr changes in pressure around the bubble are record ed. Bubble si ze ca n be adjusted with the horizontal micrometer syringe. Vertic a l syringes are for cleaning the chamber.
and of the bubble radius ( r), the surface tension was calculated using the formula of Laplace: 1::.
P
=
2 yjr.
It should be noted that in this formula 1::. P is in dynes per square centimeter, and, therefore, the value read from the record in centimeters of water had to be multiplied by 980. The radius is in centimeters, and the value measured in p. therefore had to be multiplied by 10- 4 • Bubble stability studied with communicating chambers. Two test chambers with glass
capillaries as previously described, each containing the sample, could be made to communicate individually or together with a micrometer syringe and with a pulsator unit. With the stopcock in position I (Fig. 3 ) , the micrometer syringe communicated with the chamber to the right and a bubble with a diameter of about 1,000 p. was formed in this chamber. The stopcock was then turned to position II and a bubble of equal size was formed in the left chamber. After waiting 1, 2, and 3 minutes, respectively, the stopcock was turned to position III and the pulsator unit was started. If the test chambers con-
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Sooner or later one bubble decreased in size and the time for complete disappearance was noted. This time varied even when experimental conditions were identical. Each determination was therefore done in triplicate and a mean obtained (Fig. 4). Results
y Fig. 3. Bubble stability studied with commumcating chambers . With stopcock in position I and II a bubble is formed in each chamber. After 1, 2, or 3 minutes the chambers are made to communicate by turning stopcock to position III.
In each of the lambs studied there was a flow of liquid from the trachea. The flow ranged from 0.026 to 0.13 mi. per kilogram per minute. In the lamb of the unanesthetized ewe, pH and pC0 2 from the femoral artery ranged from 7.27 to 7.37 and from 39 to 42 mm. Hg during the 4 hour period of sampling. Surface tension as it affects resistance to the first breath. The modified maximal bubble pressure method gave a surface tension for tracheal fluid of 68 ± 5 dynes per centimeter (mean ± S.D.). The values ranged from 59 to 72 dynes per centimeter (Table I) . Surface properties of a bubble. Values of surface tension in a nonpulsating bubble blown in tracheal fluid are also listed in Table I. During the 30 minutes in which a bubble was studied, surface tension de-
:I
:!i...
~
Bubble
---+-. . . . . -2l--..---
on Saline
-4-
-+-........:
o -3J .....
'mm Fig. 4. With tracheal fluid in the communicating test chambers (Fig. 3), the bubbles pulsate in parallel. One bubble slowly decreases in size and finally disappears while the other becomes correspondingly larger. With water in the test chambers the change from A to D is instantant>ous.
! -ll w -2
0: ::0
~
-3
~-+-........!:'
Bubb le in Tracheal Fluid
0: 0.
Max imal
bubb le
tained a pure liquid such as water one bubble immediately disappeared and the other became correspondingly larger. However, with tracheal fluid in the chambers the two bubbles pulsated in parallel for a time.
S1U
Fig. 5. Tracing of chamber pressure when the bubble is pulsating in water and when it has been pulsating in tracheal fluid 1, 10, and 30 minutes. Note the increase in amplitude with aging.
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Table I. Surface tension of tracheal fluid as determined with maximal bubble pressure method and as calculated from pressure surrounding a nonpulsating· bubble ~-----------------
Lamb No.
I
Weight (Kg.)
1
2.8
2
1.4
3
1.5
4
4.0
5 6
2.6 3.9
Mean± S.D.
------------------
Surface tension in dynes per centimeter 1-----------.-----Maximum bubble In nonpulsating bubble after (min.) pressure method 2 Yo I I
---~-------·--
72 69 59 66 70
72 68 ± 5
creased, and the lowest values were those measured at the end of this period. The mean was 46 ± 8 dynes per centimeter and the values ranged from 37 to 60 dynes per centimeter. The highest value, 60 dynes per centimeter, was found in the tracheal fluid of lamb No. 1, the mother of whom had been asphyxiated prior to the laparotomy. Recording of pressure around a pulsating bubble gave characteristic tracings (Fig. 5). With saline solution in the chamber, the pressure at maximal and minimal size of the bubble (radius, 500 fL and 420 fL, respectively) was what one would expect from the formula of Laplace, assuming a consistent surface tension of 72 dynes per centimeter. The _greatest negative pressure was noted at the time when the bubble was of minimal size. When the bubble was pulsating in tracheal fluid, however, the pattern of the pressure tracing was immediately different and characteristically changed with time. The relationship between bubble size and pressure was reversed so that with tracheal fluid in the chamber the greatest negative pressure was noted at a time when the bubble was of maximal size. Therefore, the calculated values for surface tension were highest when the bubble was largest and lowest when it was smallest, i.e., when its surface area was maximally compressed. As seen from Table II and Fig. 6 surface tension of the minimal size bubble decreased considerably during the 30 minutes of recording. This was particu-
69 55 5:! 55 61 5:> 57.5 :': 7
70
59 53 56 6~
54 59:': 6
66
60
53 51 53 59
37 44 4" .}
47
51
+7
55.5 :': 6
46 :' 8
• Maximal bubble size
70
60
o Minimal bubble size
jIii I
'"'
40
Q)
,..,c
Cl
30
I
l
50
E
T
T
llq
0
:w
Rill l t~ I
--1--1-t
n~ol~oI I
20
l
T
I
1
i
i
o
1
1
'
'
I
l
10
I
1
~--~----~
5
10
15
20
25
30
Minutes
Fig. 6. Change of maximal and minimal surface tl"nsion of tracheal fluid with time. Mean :': standard deviation.
larly true with the tracheal fluids from Iambs No. 2 through 6. Bubble stability in communicating chambers. The time for one of the two bubbles to disappear, after communication had been established between the chambers, was determined in triplicate and the mean values are presented in Table III. While the bubbles pulsated in parallel, one of them increased slowly in size. The other became correspondingly smaller until it disappeared. When its diameter was only a fourth to a fifth of
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Table II. Maximal and minimal apparent surface tension (dynes per centimeter) of tracheal fluid in a pulsating bubble. Surface area compressed to 70 per cent
I MfnI MTnI Minutes utes utes
IMfnutes
10 Min- 115 Minutes utes
size
Minute
Maximum Minimum
72 53
71 51
70 50
70 49
70 48
66 46
2
Maximum Minimum
59 43
56 41
56 39
56 38
55 37
3
Maximum Minimum
56 42
55 40
55 38
55 37
4
Maximum Minimum
59 42
56 38
55 31
Maximum Minimum
64 47
64 46
Maximum Minimum
61 43
58 41
Lamb No.
5 6 Mean :t S.D.
Bubble
Maximum Minimum
utes
utes
utes
66 45
65 44
65 44
65 44
52 31
53 25
53 22
53 15
52 15
54 36
53 31
51 27
51 25
51 21
51 19
55 23
54 22
53 17
53 14
53 13
53 13
53
64 45
62 45
62 44
59
55 25
55 20
55
3:1
16
53 17
56 38
55 36
54 34
51 25
52 25
53
54 15
54 15
62:!: 6 60±6 59±6 59± 6 58± 7 45 ±4 43 ± 5 40± 6 38 ± 9 37 ± 9
Table III. Time during which bubbles pulsated in parallel in communicating chambers Chambers separated (min.)
Tracheal fluid from lamb No.
1
2
(sec.)
(sec.)
7
5
20 10 29 16
14 18 17 30 16
6 Mean
14 16
16 19
1 2 3 4
I Min20 I Min25 I Min30
3 (sec.)
7
67 39 260 44 64 80
what it had been, it sometimes remained at this size for 60 to 90 seconds. Even during this time it continued to pulsate but contributed very much less to the exchange of air than did the larger bubble. Tracheal fluid from lamb No. 1 seemed to yield the most unstable bubbles and the pressure recordings pointed to a difference between this fluid and the other samples. Its surface tension never decreased to the low values observed in each of the other fluids at minimal bubble size (Table II). Comment
Under the experimental conditions of this study there was a steady flow of fluid from
17
13
56± 6 55±6 55± 5 55± 5 55±5 31±1027±10 24 ± 11 21 ± 11 21 ± 12
the trachea of the fetal lamb as previously described by Adams and associatesY· 15 The lungs may thus contribute to the formation of amniotic fluid, a finding which certainly deserves further investigation. It was anticipated that, with a collecting system such as described (Fig. 1 ) , there would be the same difference in pressure between the trachea and the collecting container as between the trachea and the amniotic cavity into which the fluid presumably flows. Furthermore, when the rubber container was emptied with suction through the narrow tubing, it would when empty, be sucked to the opening of this tubing and thereby make it impossible to produce a negative pressure in the trachea. In this study two different methods were used for determining surface tension. This was found necessary since in the newborn infant there are at least two important aspects to the problem of this physical phenomenon. When the first breaths are taken by the newborn, air is rapidly sucked into the thousands of capillary size airway branches. These were previously filled with liquid, the surface tension of which offers resistance in the hemispherical air-liquid interfaces. This
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capillarity cannot be lowered by a surfactant requiring time for "aging." Therefore, mt~th ods such as the Wilhelmy balance technique which give ample time for surfactant to become concentrated in the surface, are likely to give values of surface tension lower than those affecting initial aeration. It seems mort> likely that the latter values would be obtained with a method which directly determines capillarity, as does the maximal buhblt> pressure method used in this study. The principal of the maximal bubble prt>ssure method has been used in two previous studies on surface tension resistance to initial aeration:'· 17 It was found that amniotic fluid from humans and guinea pigs has a surface tension almost as high as that of water. In mature guinea pig fetuses it was also high in the liquid obtained from the trachea but that obtained from the main bronchi was significantly lower. The high values of fetal lamb tracheal fluid surface tension obtained with the maximal bubble pressure method correspond to those found in the trachea of guinea pig fetuses. If the lamb has a low surface tension in the fluid of its main bronchi as does the guinea pig. it must be assumed that this fluid loses some of its surface activity as it moves into tlw trachea. It could do so by becoming diluted or by becoming contaminated with counteracting agents, possibly produced by the many mucous glands emptying into the trachea."' Surface tension of tracheal fluid thus seems to offer great resistance to initial aeration. and the surfactants present probably do not have enough time to facilitate it. Once the alveoli have becomt> aerated however, a second and equally important aspect comes into play, that which has to do with alveolar stability. The maximal bubble pressure method sheds no li,g-ht on this part of the problem but the second method used in this study for determining surface tension (i.e., to measure pressure around a bubble pulsating in the sample liquid) was quite infom1ative. When a bubble was allowed to remain at a certain size and surfactant was thus given time to become concentrated in the air-
,luw 1·1 \!JI
{)h.., I
8.:
l!Jb:t
f~~'fli•C
liquid interface. sttrface tension decn•ased (Table I). After 30 minutes the mean \ altw of surface tension in a nonpulsating htJhblc was 46 dynes per centimet<·r. and it w;h around this valur the surface tension o~cil lated in the air-liquid interfan· of tht~ ptllsating bubblr. It appeared as though trw s•trfactant had formed a layer which r<'jcctl'd any change in bubble size. Tn enlarg•· til(' bubble, a gTeater negatiw pn·ssure had to he exerted than would he rrquired to 0\t'l·· come the mean surface tension alone. \Yhf'll the bubble was made to decrease in size. tfw surface laver seemed also to counteract such a chant!,"C, whereby the bubble needed ]pss and less negative pressure to rt>main expanded. With this concept in l!lincl it is r·as\ to comprehend the stabilizing ability of tlw surfactant. The bubbles in the conmlllnicating chambers continued to pulsate in parallt-1 for a certain time because their Sl!lfa(T layers rejected any chang·e in buhhk ;;i;t· When t>\·cntually one bubble did hecorne \1'1' smalL for a while its surface layer m~utrali;ed the tendency of surface tension to make tlw bubble shrink further. ThP surface 1ens ion at that time must have heen clost• to ;en> in the small bubble. Pattk"· 7 evaluated lung surface propertiq, by watching under the microscope how Jon!.! it takt's for the gas in bubbles expressed from lung tissues to become absorbed hy the liqllicl surrounding the bubbles. Ht> came to tlw conclusion that the longt>r the bubbles <·Xi'-!. the lowt'r is the surface' tension in tfwir airliquid intt'rfacc. Bubblt's expressed from normal lungs existed for such a long time that Pattie believed that surface tension was dos" to zt>ro. The hubblt's, when 11rst immersed into ttH' liquid. must han· been larger, b1Jt as some of their gas dissolved into th1• Sllrrounding liquid, they decreased in si;c allCI their surfactants rejected further compression of the surface layer, giving an apparent surface tension of zero. However. as demonstrated by Langmuir and Schadler"" there is ~' rt>sistanC!' to diffusion throu~·h com]nes,t·d surface layers, and this would also tend to pro!on~· the lifetime of the shrunken buhbk. If indeed the surface tension decreases rn
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Surface properties of fetal lamb tracheal fluid
almost zero it will only occur under conditions of extreme compression. Recording of pressure around a bubble pulsating in tracheal fluid has given results which are in agree1nent with the important finding of Langmuir 21 that surface tension decreases when the surface area is compressed. However, the minimal values recorded with maximal compression of surface area were not so low as those found in a study with the modified Wilhelmy balance. 22 This is probably mainly due to the fact that in the trough of the balance the surface area is given more time to age. Furthermore, the trough surface is compressed to 20 per cent of its maximal size, whereas the bubble surface \vas only reduced to 70 per cent of its maximal size. This amount of compression would seem closer to the change that may occur in the alveolar surface during breathing. Calculations show that when young men t:>xhale maximally the surface area is compressed to about 35 per cent of its maximal size, and with normal breathing it is compressed to only about 85 per cent. The findings of the present study are in agreement with the concept of Clements and co-workers 9 that for alveolar stability surface tension dccn·~.<;p - - - - - - - - -h~s - - - - -to ---- - - - - - - - rlurinp· ------o comnrcssion of surface area. As judged from this, the pressure recordings demonstrate that during the first 30 minutes the bubble becomes increasingly stable and the experiments with the communicating chambers also imply that stability increases with time. During the first seconds or minutes after initial aeration, stability may still be inadequate and the speed with which the surface layer develops would seem to be of vital importance for the <'stablishment of normal extrauterine life. The tracheal fluid from lamb No. I, the mother of whom had been seriously distressed prior to the laparotomy, was quite different from the other samples. Its surface tension decreased only slightly, and after 30 minutes was still 60 dynes per centimeter. After 30 minutes of pulsating, the minimal value of apparent surface tension was still 44 dynes ~---
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per centimeter or appr0%imately three times more than any of the corresponding values of the other samples. In the communicating chambers, this tracheal fluid also yielded the most unstable bubbles. The cause of this difference is not known. If it had anything to do with the asphyxia of the ewe, a possible explanation would be that the lamb had aspirated amniotic fluid repeatedly becaus<~ or mtrautenne mstress ana tnereoy wasneu out the liquid normally present in the airways. Further research would seem indicated to determine if in this way a period of intrauterine distress may be the cause of a more difficult establishment of extrauterine respiration. I"
•
•
•
•
,.
'
1
.1
1
1
1
Summary
A liquid flowing at a rate of 0.026 to 0. U mi. per kilogram per minute from the trachea of 6 lambs in utero was collected for a study of its surface properties by three methods. 1. With a modified maximal bubble pressure method, it was found that the surface tension affecting initial aeration was very high, 68 ± 5 dynes per centimeter (mean ±S.D.). 2. By recording pressure around a bubble in tracheal fluid, it was found that surface tension decreased with time. After 30 minutes it was 46 ± 8 dynes per centimeter. When the bubble was made to pulsate in this chamber, its surface tension oscillated around this value. When the bubble was of minimal size, and its surface area therefore compressed, the surface tension after 30 minutes of pulsating was 21 ± 12 dynes pn centimeter. The corresponding value at maximal bubble size was 55 ± 5 dynes per centimeter. Tracheal fluid thus contains a surfactant, which may stabilize the alveoli. 3. The stabilizing ability of the surfactant was more directly demonstrated with two bubbles in communicating chambers. When the latter contained tracheal fluid the two bubbles pulsated in parallel for up to 4 minutes.
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REFERENCES
1. Wilson, ]. L., and Farbert, S.: Am. ]. Dis. Child. 46: 590, 1933. 2. Karlberg, P., Cherry, R. B., Escardo, F. E., and Koch, G.: Acta paediat. 51: 212, 1962. 3. Gruenwald, P.: AM. ]. 0BST. & GYNEC. 53: 996, 1947. 4. Agostoni, E., Taglirtti, A., Agostoni, A. F., and Setnikar, 1.: ]. Appl. Physiol. 13: 344, 1958. 5. Enhi:irning, G., and Kirschbaum, T. H.: Al'
13. Avery. M. E., and Mead, ].: Am. ]. Dis. Chile!. 97: 517, 1959. 14. Adams, F. H., Moss, A . .)., and Fagan, L.: Bioi. Neonat. 5: 151, 1963. 15. Adams, F. H., Fujiwara, T., and Rowshan, G.: ]. Pediat. 63: 881, 1963. 16. Astrup, P., Jorgensen, K., Andersen, 0 S.. and Engel, K.: Lancet 1: 1035, 1960. 17. Enhi:irning, G.: AM. ]. 0BsT. & GYNEC. ll8: 519, 1964. 18. Enhi:irning, G. Adams, F. H., and Scudder, A.: To be published. 19. Ham, A. W., and Leeson, T. S.: Histology, ed. 4, Philadelphia, 1961. J. B. Lippincott Company, p. 670. 20. Langmuir, I.. and Schaeffer, V. ]. : In Moulton, F. R., editor: Surface Chemistry, \'llashington, D. C., 1943, Publication of the American Association for the Advancement of Science. No. 21, p. 17. 21. Langmuir, I.: ]. Am. Chern. Soc. 39: 18·HL 1917. 22. Adams, F. H., and Fujiwara, T.: .J. Pediat. 63: 537, 1963.
F r R s T breaths of the newborn infant are the most difficult. 1 Intraesophageal pressure recordings indicate that they offer much greater resistance than subsequent inspirations.2 Prim· to the first breath the trachea and the finer branches of the airvvays arc filled with liquid. When the intrathoracic pressure is lowered in an attempt to aerate the lungs, this liquid has to move ahead of the air and its viscosity and surface tension is an obstacle to these first breaths. Few studies have been focused on the resistance that surface tension of the airway liquid offers to the first breath. 3 - 5 More interest has been given to its importance once respiration has been established. 6 ' 12 The alveolus will close with exponential speed if the surface tension of its lining layer remains at a fixed value. There is good evidence, however, that surface tension decreases as the alveolus becomes smaller and the tendency of the alveolus to close is lessened. 8 - 13 There is also good evidence that this ability to stabilize the alveoli by a substantial decrease of surface tension with decreased alveolar size is absent or inadequate in newborns dying of the respiratory distress syndrome.13 One of the major problems of studying this aspect of surface tension lies in the difficulty of obtaining a sample of the liquid in the air-liquid interface of the alveoli. Usually
the whole lung is cut into small pieces and stirred with physiological saline solution. This gives an extract of which 50 ml. is required for a study with the modified Wilhelmy balance. 9 ..A1dams and co-\vorkers 14 ' 1 u have desciibed a flow of liquid from the trachea in fetal lambs. At the time of the first breath this liquid must be pulled into the alveoli where it participates in forming a noncellular layer. If it has the ability of lowering surface tension with decreasing alveolar size, it will help to stabilize the alveoli. In this study, the surface properties of tracheal fluid collected from lambs in utero were evaluated. The surface tension, as it may affect resistance to the first breath was determined. The apparent changes in surface tension, which may occur during respiration, were recorded with a method which simulates alveolar ventilation. The alveolar stabilizing property of the surface layer was evaluated from these recordings and was also studied with a more direct approach.
THE
Materials and methods Collection of tracheal fluid. Six lambs from 6 ewes were used for this study. Five of the ewes were anesthetized with pentobarbital sodium injected into the jugular vein which had been dissected free under local anesthesia. An initial dose of from 0.5 to 0.65 Gm. was administered and supplemental doses of 0.5 Gm. were given as needed. A tracheotomy was done so that 100 per cent oxygen could be supplied to the ewe. A Harvard respirator was used. Its stroke volume was set at 350 mi. and its frequency at 24 to 28 strokes per minute. The blood pressure
From the Department of Pediatrics, School of Medicine, University of California, Los Angeles. Supported in Part by grants from United Cerebral Palsy Research and Educational Foundation (Dwight D. Eisenhower Fund) and the United States Public Health Service.
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was monitored during the operation and was consistently found to be within normal values. Ten minutes after the respirator was started, a blood sample was taken from the carotid artery for pH and pC0 2 determinations by the micromethod of Astrup and associates.1'; The values were within the normal range* in 5 sheep, but in one ewe, the one with Iamb No. 1 of this study, a decreasing pH and an increasing pC0 2 indicated inadequate ventilation. The cause was found and eliminated but in this ewe there had thus been a short period of severe asphyxia in the initial part of the experiment. During the operation the ewe was lying on her right side. The abdominal wall was incised in the midline and the pregnant uterine horn marsupialized to the edge of the abdominal wound. The uterus was then opened and the head of the Iamb was delivered. The trachea was exposed, and when it was opened just below the larynx, clear fluid poured out under pressure. A special container was used for its collection. It consisted of a rubber glove finger threaded over and tightly secured to the ends of two pieces of vinyl tubing. One tubing was short and wide (length, 10 em.; outer diameter, 0.5 em.), and when it had been introduced into the trachea and tightly secured, it gave free outlet to the rubber container. This container could be emptied intermittently through the other tubing which was long and narrow (length, 50 em.; outer diameter, 0.2 em.). After the tracheotomy and the introduction of the wide tube into the trachea, the head of the lamb as well as the rubber container were returned into the uterus which was closed, as was the abdominal wall. However, the narrow tubing formed an outlet through the uterine and abdominal incisions (Fig. I). In one ewe, the mother of lamb No. 5 of this study, the operative procedure was slightly different. In this animal a total of 1.3 Gm. of pentobarbital sodium was injected into one of the dilated abdominal veins. Without doing· a tracheotomy or using *Normal values in the unanesthetized, standing, term ewe for our laboratory range as follows: pH = 7.41 to 7.48; pCO, = 29 to 34 mm. Hg.
J. Obst.
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the respirator, the operation was performed as described above. Howe\·er, in this case a hind leg was also delivered so that a catheter could be introduced into the femoral artcrv. This catheter together with the tubing from the collecting rubber container led through the abdominal wall. When the latter had been closed the ewe was allowed to wake up. Within an hour she was standing and che11ing her cud. Samples of tracheal fluid and blood for pH and pC0 2 determinations were thus obtained from the fetus while the f'WP was unanesthetized. Determination of surface tension as it affects resistance to the first breath. A modification of the maximum bubble pressure method was used. The technique has previously been described in a study on amniotic fluid 1 ; and therefore only its principal i'i outlined. Two small chambers, one containing water, the other thP sample, communicated with open air via fine bore n'ttical glass capillaries which had identical dimensions at the tip, inside the chamber. The capillaries contained liquid but as the chambers were slowly evacuated one at a time. a meniscus mo\'ed towards the capillary tip. The chambt'r was connected to a pressun~ transducer which recorded t}w negatin~ pressure which came to a peak ( ~ P) and became less negative as air mC)\'Cd to the capillary tip and a bubble was suddenly formed. Surface tension ( Ys I of the sample was calculated from the pressure dett'rmination since it is proportional to the surface tension of water ( Yw) as the peak pressure in the sample chamber (:l P,l is proportional to that in the water chamber ( ~ Pw) ; i.e.: y s
t. P,
y w
t. 1\,.
from which Ys was calculated since Yw is 72 dynes per centimeter at 20° C. Determination of surface properties of a bubble in tracheal fluid. A model of an alveolus and its airway was constructed which simulated the respiratory changes in akeolar size such that the surface properties could be evaluated. As shown in Fig. 2, this consisted of a chamber for the sample fluid which
Volume 92 N umber 4
Surface properties of fetal lamb tracheal fluid 565
Fig. 1. Method for collec ting fetal lamb tracheal fluid in utero.
communicated with open air via a vertical glass capillary, the "airway." The chamber also communicated with a micrometer syringe, a pressure transducer and a pulsator unit. It was originally filled with the sample fluid, but when some of this was withdrawn with the micrometer syringe, air was sucked into the capillary and a bubble, the "alveolus," was formed in the chamber. At a frequency of 17 cycles per minute the pulsator moved 0.21 mm. 3 of liquid in and out of the chamber. This gave a cyclic change in bubble size. Each time the bubble was of minimal size, a mark was automatically made on the pressure record, and it was known that the maximal bubble size was exactly half way between two moments of minimal size, thus, half way between two marks. The pressure chamber was mounted on the objective table of a microscope and was moved in relation to the latter so that the op tical axis of th e microscope went through the center of the bubble. In this way the diameter of the bubble could be measured with a scale in the eye-piece of the microscope. A detailed description of the method is published elsewhere. 18 The chamber was fill ed with physiological saline solution and calibrated at -2 and -4 em. of water. By withdrawing with the
micrometer syringe a bubble was formed at the tip of the capillary. As the pulsator unit was started, the micrometer syringe was adjusted so that the maximal diameter of the bubble was 1,000 f.L· The minimal diameter was then ap proximately 840 p.. When the chamber contained physiological saline solution, this cyclic change in bubble size gave a characteristic symmetrical pressure tracingdemonstrating that the sample chamber had been adequately cleaned. The chamber was then calibrated at -1 and -3 em. of water and the physiological saline solution replaced with a sample of tracheal fluid . Again a bubble with a diameter of 1,000 p. was made at the capillary tip and the bubble was kept at this size while the pressure was recorded for a few minutes. The bubble was then extruded and replaced with a new one. The pulsations were started and continued for 30 minutes while the pressure was continuously recorded. Adjustments with the micrometer syringe secured a maximal bubble diameter of 1,000 f.t. When the pulsator unit was stopped, the bubble diameter was kept stable at 1,000 p. for approximately 2 minutes. The calibration was controlled at the end of each determination. With knowledge of the pressure gradient (~ P ) required to keep the bubble expanded,
566
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:\m .
.1.
.I Hill' h . I ~~b Uhst. & Gyncc
.1
lJ
ACriiAL PRESSIJREIIECOROOF WArEIIBIJBBLE
Fig. 2. Principle (a) and arrangement (b) for studying surface properties of a bubble in a liquid. The bubble communicates with atmosphere via a glass capillary and its size is determined with a scale in the eyepiece of the microscope. The bubble becomes large r and smaller as th e pulsator unit moves a small amount of liquid in and out of the chamber, simulating relative changes in alveolar size during respiration. Thr changes in pressure around the bubble are record ed. Bubble si ze ca n be adjusted with the horizontal micrometer syringe. Vertic a l syringes are for cleaning the chamber.
and of the bubble radius ( r), the surface tension was calculated using the formula of Laplace: 1::.
P
=
2 yjr.
It should be noted that in this formula 1::. P is in dynes per square centimeter, and, therefore, the value read from the record in centimeters of water had to be multiplied by 980. The radius is in centimeters, and the value measured in p. therefore had to be multiplied by 10- 4 • Bubble stability studied with communicating chambers. Two test chambers with glass
capillaries as previously described, each containing the sample, could be made to communicate individually or together with a micrometer syringe and with a pulsator unit. With the stopcock in position I (Fig. 3 ) , the micrometer syringe communicated with the chamber to the right and a bubble with a diameter of about 1,000 p. was formed in this chamber. The stopcock was then turned to position II and a bubble of equal size was formed in the left chamber. After waiting 1, 2, and 3 minutes, respectively, the stopcock was turned to position III and the pulsator unit was started. If the test chambers con-
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Surface properties of fetal lamb tracheal fluid
567
Sooner or later one bubble decreased in size and the time for complete disappearance was noted. This time varied even when experimental conditions were identical. Each determination was therefore done in triplicate and a mean obtained (Fig. 4). Results
y Fig. 3. Bubble stability studied with commumcating chambers . With stopcock in position I and II a bubble is formed in each chamber. After 1, 2, or 3 minutes the chambers are made to communicate by turning stopcock to position III.
In each of the lambs studied there was a flow of liquid from the trachea. The flow ranged from 0.026 to 0.13 mi. per kilogram per minute. In the lamb of the unanesthetized ewe, pH and pC0 2 from the femoral artery ranged from 7.27 to 7.37 and from 39 to 42 mm. Hg during the 4 hour period of sampling. Surface tension as it affects resistance to the first breath. The modified maximal bubble pressure method gave a surface tension for tracheal fluid of 68 ± 5 dynes per centimeter (mean ± S.D.). The values ranged from 59 to 72 dynes per centimeter (Table I) . Surface properties of a bubble. Values of surface tension in a nonpulsating bubble blown in tracheal fluid are also listed in Table I. During the 30 minutes in which a bubble was studied, surface tension de-
:I
:!i...
~
Bubble
---+-. . . . . -2l--..---
on Saline
-4-
-+-........:
o -3J .....
'mm Fig. 4. With tracheal fluid in the communicating test chambers (Fig. 3), the bubbles pulsate in parallel. One bubble slowly decreases in size and finally disappears while the other becomes correspondingly larger. With water in the test chambers the change from A to D is instantant>ous.
! -ll w -2
0: ::0
~
-3
~-+-........!:'
Bubb le in Tracheal Fluid
0: 0.
Max imal
bubb le
tained a pure liquid such as water one bubble immediately disappeared and the other became correspondingly larger. However, with tracheal fluid in the chambers the two bubbles pulsated in parallel for a time.
S1U
Fig. 5. Tracing of chamber pressure when the bubble is pulsating in water and when it has been pulsating in tracheal fluid 1, 10, and 30 minutes. Note the increase in amplitude with aging.
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Table I. Surface tension of tracheal fluid as determined with maximal bubble pressure method and as calculated from pressure surrounding a nonpulsating· bubble ~-----------------
Lamb No.
I
Weight (Kg.)
1
2.8
2
1.4
3
1.5
4
4.0
5 6
2.6 3.9
Mean± S.D.
------------------
Surface tension in dynes per centimeter 1-----------.-----Maximum bubble In nonpulsating bubble after (min.) pressure method 2 Yo I I
---~-------·--
72 69 59 66 70
72 68 ± 5
creased, and the lowest values were those measured at the end of this period. The mean was 46 ± 8 dynes per centimeter and the values ranged from 37 to 60 dynes per centimeter. The highest value, 60 dynes per centimeter, was found in the tracheal fluid of lamb No. 1, the mother of whom had been asphyxiated prior to the laparotomy. Recording of pressure around a pulsating bubble gave characteristic tracings (Fig. 5). With saline solution in the chamber, the pressure at maximal and minimal size of the bubble (radius, 500 fL and 420 fL, respectively) was what one would expect from the formula of Laplace, assuming a consistent surface tension of 72 dynes per centimeter. The _greatest negative pressure was noted at the time when the bubble was of minimal size. When the bubble was pulsating in tracheal fluid, however, the pattern of the pressure tracing was immediately different and characteristically changed with time. The relationship between bubble size and pressure was reversed so that with tracheal fluid in the chamber the greatest negative pressure was noted at a time when the bubble was of maximal size. Therefore, the calculated values for surface tension were highest when the bubble was largest and lowest when it was smallest, i.e., when its surface area was maximally compressed. As seen from Table II and Fig. 6 surface tension of the minimal size bubble decreased considerably during the 30 minutes of recording. This was particu-
69 55 5:! 55 61 5:> 57.5 :': 7
70
59 53 56 6~
54 59:': 6
66
60
53 51 53 59
37 44 4" .}
47
51
+7
55.5 :': 6
46 :' 8
• Maximal bubble size
70
60
o Minimal bubble size
jIii I
'"'
40
Q)
,..,c
Cl
30
I
l
50
E
T
T
llq
0
:w
Rill l t~ I
--1--1-t
n~ol~oI I
20
l
T
I
1
i
i
o
1
1
'
'
I
l
10
I
1
~--~----~
5
10
15
20
25
30
Minutes
Fig. 6. Change of maximal and minimal surface tl"nsion of tracheal fluid with time. Mean :': standard deviation.
larly true with the tracheal fluids from Iambs No. 2 through 6. Bubble stability in communicating chambers. The time for one of the two bubbles to disappear, after communication had been established between the chambers, was determined in triplicate and the mean values are presented in Table III. While the bubbles pulsated in parallel, one of them increased slowly in size. The other became correspondingly smaller until it disappeared. When its diameter was only a fourth to a fifth of
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569
Table II. Maximal and minimal apparent surface tension (dynes per centimeter) of tracheal fluid in a pulsating bubble. Surface area compressed to 70 per cent
I MfnI MTnI Minutes utes utes
IMfnutes
10 Min- 115 Minutes utes
size
Minute
Maximum Minimum
72 53
71 51
70 50
70 49
70 48
66 46
2
Maximum Minimum
59 43
56 41
56 39
56 38
55 37
3
Maximum Minimum
56 42
55 40
55 38
55 37
4
Maximum Minimum
59 42
56 38
55 31
Maximum Minimum
64 47
64 46
Maximum Minimum
61 43
58 41
Lamb No.
5 6 Mean :t S.D.
Bubble
Maximum Minimum
utes
utes
utes
66 45
65 44
65 44
65 44
52 31
53 25
53 22
53 15
52 15
54 36
53 31
51 27
51 25
51 21
51 19
55 23
54 22
53 17
53 14
53 13
53 13
53
64 45
62 45
62 44
59
55 25
55 20
55
3:1
16
53 17
56 38
55 36
54 34
51 25
52 25
53
54 15
54 15
62:!: 6 60±6 59±6 59± 6 58± 7 45 ±4 43 ± 5 40± 6 38 ± 9 37 ± 9
Table III. Time during which bubbles pulsated in parallel in communicating chambers Chambers separated (min.)
Tracheal fluid from lamb No.
1
2
(sec.)
(sec.)
7
5
20 10 29 16
14 18 17 30 16
6 Mean
14 16
16 19
1 2 3 4
I Min20 I Min25 I Min30
3 (sec.)
7
67 39 260 44 64 80
what it had been, it sometimes remained at this size for 60 to 90 seconds. Even during this time it continued to pulsate but contributed very much less to the exchange of air than did the larger bubble. Tracheal fluid from lamb No. 1 seemed to yield the most unstable bubbles and the pressure recordings pointed to a difference between this fluid and the other samples. Its surface tension never decreased to the low values observed in each of the other fluids at minimal bubble size (Table II). Comment
Under the experimental conditions of this study there was a steady flow of fluid from
17
13
56± 6 55±6 55± 5 55± 5 55±5 31±1027±10 24 ± 11 21 ± 11 21 ± 12
the trachea of the fetal lamb as previously described by Adams and associatesY· 15 The lungs may thus contribute to the formation of amniotic fluid, a finding which certainly deserves further investigation. It was anticipated that, with a collecting system such as described (Fig. 1 ) , there would be the same difference in pressure between the trachea and the collecting container as between the trachea and the amniotic cavity into which the fluid presumably flows. Furthermore, when the rubber container was emptied with suction through the narrow tubing, it would when empty, be sucked to the opening of this tubing and thereby make it impossible to produce a negative pressure in the trachea. In this study two different methods were used for determining surface tension. This was found necessary since in the newborn infant there are at least two important aspects to the problem of this physical phenomenon. When the first breaths are taken by the newborn, air is rapidly sucked into the thousands of capillary size airway branches. These were previously filled with liquid, the surface tension of which offers resistance in the hemispherical air-liquid interfaces. This
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Enhorning and Adams
capillarity cannot be lowered by a surfactant requiring time for "aging." Therefore, mt~th ods such as the Wilhelmy balance technique which give ample time for surfactant to become concentrated in the surface, are likely to give values of surface tension lower than those affecting initial aeration. It seems mort> likely that the latter values would be obtained with a method which directly determines capillarity, as does the maximal buhblt> pressure method used in this study. The principal of the maximal bubble prt>ssure method has been used in two previous studies on surface tension resistance to initial aeration:'· 17 It was found that amniotic fluid from humans and guinea pigs has a surface tension almost as high as that of water. In mature guinea pig fetuses it was also high in the liquid obtained from the trachea but that obtained from the main bronchi was significantly lower. The high values of fetal lamb tracheal fluid surface tension obtained with the maximal bubble pressure method correspond to those found in the trachea of guinea pig fetuses. If the lamb has a low surface tension in the fluid of its main bronchi as does the guinea pig. it must be assumed that this fluid loses some of its surface activity as it moves into tlw trachea. It could do so by becoming diluted or by becoming contaminated with counteracting agents, possibly produced by the many mucous glands emptying into the trachea."' Surface tension of tracheal fluid thus seems to offer great resistance to initial aeration. and the surfactants present probably do not have enough time to facilitate it. Once the alveoli have becomt> aerated however, a second and equally important aspect comes into play, that which has to do with alveolar stability. The maximal bubble pressure method sheds no li,g-ht on this part of the problem but the second method used in this study for determining surface tension (i.e., to measure pressure around a bubble pulsating in the sample liquid) was quite infom1ative. When a bubble was allowed to remain at a certain size and surfactant was thus given time to become concentrated in the air-
,luw 1·1 \!JI
{)h.., I
8.:
l!Jb:t
f~~'fli•C
liquid interface. sttrface tension decn•ased (Table I). After 30 minutes the mean \ altw of surface tension in a nonpulsating htJhblc was 46 dynes per centimet<·r. and it w;h around this valur the surface tension o~cil lated in the air-liquid interfan· of tht~ ptllsating bubblr. It appeared as though trw s•trfactant had formed a layer which r<'jcctl'd any change in bubble size. Tn enlarg•· til(' bubble, a gTeater negatiw pn·ssure had to he exerted than would he rrquired to 0\t'l·· come the mean surface tension alone. \Yhf'll the bubble was made to decrease in size. tfw surface laver seemed also to counteract such a chant!,"C, whereby the bubble needed ]pss and less negative pressure to rt>main expanded. With this concept in l!lincl it is r·as\ to comprehend the stabilizing ability of tlw surfactant. The bubbles in the conmlllnicating chambers continued to pulsate in parallt-1 for a certain time because their Sl!lfa(T layers rejected any chang·e in buhhk ;;i;t· When t>\·cntually one bubble did hecorne \1'1' smalL for a while its surface layer m~utrali;ed the tendency of surface tension to make tlw bubble shrink further. ThP surface 1ens ion at that time must have heen clost• to ;en> in the small bubble. Pattk"· 7 evaluated lung surface propertiq, by watching under the microscope how Jon!.! it takt's for the gas in bubbles expressed from lung tissues to become absorbed hy the liqllicl surrounding the bubbles. Ht> came to tlw conclusion that the longt>r the bubbles <·Xi'-!. the lowt'r is the surface' tension in tfwir airliquid intt'rfacc. Bubblt's expressed from normal lungs existed for such a long time that Pattie believed that surface tension was dos" to zt>ro. The hubblt's, when 11rst immersed into ttH' liquid. must han· been larger, b1Jt as some of their gas dissolved into th1• Sllrrounding liquid, they decreased in si;c allCI their surfactants rejected further compression of the surface layer, giving an apparent surface tension of zero. However. as demonstrated by Langmuir and Schadler"" there is ~' rt>sistanC!' to diffusion throu~·h com]nes,t·d surface layers, and this would also tend to pro!on~· the lifetime of the shrunken buhbk. If indeed the surface tension decreases rn
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Surface properties of fetal lamb tracheal fluid
almost zero it will only occur under conditions of extreme compression. Recording of pressure around a bubble pulsating in tracheal fluid has given results which are in agree1nent with the important finding of Langmuir 21 that surface tension decreases when the surface area is compressed. However, the minimal values recorded with maximal compression of surface area were not so low as those found in a study with the modified Wilhelmy balance. 22 This is probably mainly due to the fact that in the trough of the balance the surface area is given more time to age. Furthermore, the trough surface is compressed to 20 per cent of its maximal size, whereas the bubble surface \vas only reduced to 70 per cent of its maximal size. This amount of compression would seem closer to the change that may occur in the alveolar surface during breathing. Calculations show that when young men t:>xhale maximally the surface area is compressed to about 35 per cent of its maximal size, and with normal breathing it is compressed to only about 85 per cent. The findings of the present study are in agreement with the concept of Clements and co-workers 9 that for alveolar stability surface tension dccn·~.<;p - - - - - - - - -h~s - - - - -to ---- - - - - - - - rlurinp· ------o comnrcssion of surface area. As judged from this, the pressure recordings demonstrate that during the first 30 minutes the bubble becomes increasingly stable and the experiments with the communicating chambers also imply that stability increases with time. During the first seconds or minutes after initial aeration, stability may still be inadequate and the speed with which the surface layer develops would seem to be of vital importance for the <'stablishment of normal extrauterine life. The tracheal fluid from lamb No. I, the mother of whom had been seriously distressed prior to the laparotomy, was quite different from the other samples. Its surface tension decreased only slightly, and after 30 minutes was still 60 dynes per centimeter. After 30 minutes of pulsating, the minimal value of apparent surface tension was still 44 dynes ~---
571
per centimeter or appr0%imately three times more than any of the corresponding values of the other samples. In the communicating chambers, this tracheal fluid also yielded the most unstable bubbles. The cause of this difference is not known. If it had anything to do with the asphyxia of the ewe, a possible explanation would be that the lamb had aspirated amniotic fluid repeatedly becaus<~ or mtrautenne mstress ana tnereoy wasneu out the liquid normally present in the airways. Further research would seem indicated to determine if in this way a period of intrauterine distress may be the cause of a more difficult establishment of extrauterine respiration. I"
•
•
•
•
,.
'
1
.1
1
1
1
Summary
A liquid flowing at a rate of 0.026 to 0. U mi. per kilogram per minute from the trachea of 6 lambs in utero was collected for a study of its surface properties by three methods. 1. With a modified maximal bubble pressure method, it was found that the surface tension affecting initial aeration was very high, 68 ± 5 dynes per centimeter (mean ±S.D.). 2. By recording pressure around a bubble in tracheal fluid, it was found that surface tension decreased with time. After 30 minutes it was 46 ± 8 dynes per centimeter. When the bubble was made to pulsate in this chamber, its surface tension oscillated around this value. When the bubble was of minimal size, and its surface area therefore compressed, the surface tension after 30 minutes of pulsating was 21 ± 12 dynes pn centimeter. The corresponding value at maximal bubble size was 55 ± 5 dynes per centimeter. Tracheal fluid thus contains a surfactant, which may stabilize the alveoli. 3. The stabilizing ability of the surfactant was more directly demonstrated with two bubbles in communicating chambers. When the latter contained tracheal fluid the two bubbles pulsated in parallel for up to 4 minutes.
572
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REFERENCES
1. Wilson, ]. L., and Farbert, S.: Am. ]. Dis. Child. 46: 590, 1933. 2. Karlberg, P., Cherry, R. B., Escardo, F. E., and Koch, G.: Acta paediat. 51: 212, 1962. 3. Gruenwald, P.: AM. ]. 0BST. & GYNEC. 53: 996, 1947. 4. Agostoni, E., Taglirtti, A., Agostoni, A. F., and Setnikar, 1.: ]. Appl. Physiol. 13: 344, 1958. 5. Enhi:irning, G., and Kirschbaum, T. H.: Al'
13. Avery. M. E., and Mead, ].: Am. ]. Dis. Chile!. 97: 517, 1959. 14. Adams, F. H., Moss, A . .)., and Fagan, L.: Bioi. Neonat. 5: 151, 1963. 15. Adams, F. H., Fujiwara, T., and Rowshan, G.: ]. Pediat. 63: 881, 1963. 16. Astrup, P., Jorgensen, K., Andersen, 0 S.. and Engel, K.: Lancet 1: 1035, 1960. 17. Enhi:irning, G.: AM. ]. 0BsT. & GYNEC. ll8: 519, 1964. 18. Enhi:irning, G. Adams, F. H., and Scudder, A.: To be published. 19. Ham, A. W., and Leeson, T. S.: Histology, ed. 4, Philadelphia, 1961. J. B. Lippincott Company, p. 670. 20. Langmuir, I.. and Schaeffer, V. ]. : In Moulton, F. R., editor: Surface Chemistry, \'llashington, D. C., 1943, Publication of the American Association for the Advancement of Science. No. 21, p. 17. 21. Langmuir, I.: ]. Am. Chern. Soc. 39: 18·HL 1917. 22. Adams, F. H., and Fujiwara, T.: .J. Pediat. 63: 537, 1963.