Influence of Alveolar Collapse upon Surface Activity of Lung Extracts

Influence of Alveolar Collapse upon Surface Activity of Lung Extracts-~ ALTON

1.

SUTNICK, M.D.,*·x- LOUIS A. SOLOFF, M.D.t AND RAJINDRA S. SETHI, M.D·t

Philadelphia, Pennsylvania

T

HERE IS NOW CONSIDERABLE EVIDENCE

that destruction or inactivation of pulmonary surfactant can result in atelectatic collapse of alveoli.'" Sufficient information has been obtained in the experimental animal and in the human to provide firm grounds for the bel ie f that this surface active substance is necessary for alveolar stability and patency.3.• The influence of atelectasis itself upon the integrity or activity of surfactant has received some consideration,H but the methodology is suspect, and the results have been variable. The following experiment was planned to further evaluate this relationship, and to determine if alveolar collapse will adversely affect surfactant. MATERIAL A '0 METHODS

Thirty-seven mongrel dogs weighing 1215 Ib were used for the study. They were anesthetized with pentobarbital sodium, and right thoracotomy was performed. After carefully dissecting away the bronchial vessels, the branch bronchi to two lobes were ligated with black silk suture material. The thoracic cavity was then closed, and 300,000 un its of aqueous procaine penicillin was administered intramuscularly daily. The animals were allowed to recover from anesthesia, and subsequently were $acrificed with intravenous pentobarbital at intervals of 7-96 hours following the surgical procedure. At necropsy, the entire right lung was removed for study, using the unligated

lobes as controls for each animal. If these control lobes were found to be abnormal in any way, the entire lung was discarded and not included in the experiment. One of the collapsed lobes was maximally re-expanded with air, and the other was left airless. Sections of all three lobes (normal, collapsed and re-expanded) were removed for determination of surface forces and histologic stu d y. Histologic preparations were stained with hematoxylin and eosin. Surface activity was studied on a modified Langmuir-Wilhelmy surface film balance with the same technique used in previous studies.? Three gram samples of lung tissue were minced in saline and mixed on a magnetic stirrer for 15 minutes to extract surfactant. The extract was strained through a double thickness of gauze into the Teflon trough of the surface film balance. The surface was then alternately compressed to one-fifth of its area, and re-expanded over 15 minute cycles, after first being permitted to "age" for 30 minutes. A determination was completed when the same minimal surface tension was reached on two successive cycles. rvIaximal and minimal surface tensions in dynes/ern (y) were determined, and recorded on a Houston x- Y recorder. Stability indices were calculated according to the'formula provided by

*Supported in part by United States Public Health Service Research Grants CA-06551 and HE08595, National Institutes of Health, Bethesda. **Research Physician, The Institute for Cancer Research, Assistant Professor of Medicine, University of Pennsylvania School of Medicine. t Professor of Medicine an d Chief, Division of Cardiology, Temple University Medical Center. Presently at the Institute for Cancer Research, Fox Chase, Philadelphia. ;Resident in Thoracic Surgery, Temple University Medical Center. Presently at the Institute for Cancer Research, Fox Chase, Philadelphia.

FICURE I: Histologic section of atelectatic lung 48 hours following ligation of bronchus.

257

Disc:1ses of

SUTNICK, SOLOFF A!'
Clements el al:" S = 2 (1 max - r min)/ (1 max + 1 min) . RESULTS

The ligated lobes consistently grossly demonstrated total atelectasis. They were small, contracted, dar k red in color , and would not float in water. The microscopic appearance was that of alveolar collapse (Fig 1). The re-expanded lobes appeared grossly and histologically qui t e similar to the normal lobes (Fig 2). The alveoli appeared quite normal, and there was no evidence of pulmonary edema or hyaline ~embr~ne form~tion. There was no signifICant difference In the size and number of blood vessels in t he specimens atelectatic for longer or shorter intervals. The minimal surface tensions of the 37 control lobe extracts were consistently 10 dynes/cm or below, with a mean of 7.0 dynes/cm. These results are the same as in previously studied normal doO' and hu1 1:> man lungs. The collapsed lobes removed 18 hours or more following bronchial ligation demonstrated relativelv little surface activity, with the mean r'ninimal surface tension being 21.9 dynes/cm. At seven hours, however, they had normal surface activity (Table 1). Lower stability indices were also demonstrated in lobes atelectatic for 18 hours or longer. The re-expanded lobes demonstrated a much lower mean minimal surface tension, being 13.7 dynes/cm in those excised 18 or more hours postoperatively. Indeed, the 13 animals sacrificed 24 hours or less after

2: Histologic section of atelectatic lung fe-expanded in lIitro 4B hours following ligation of hronchus.

FIGURE

the Chesl

bronchial ligation have a mean of only 9.0 dynes/cm. Nine of these 13 specimens demonstrated normal tension-area diagrams. Those lobes collapsed for longer than 24 hours had a mean minimal surface tension of 16.5 dynes/em, in contrast to 7.0 dynes/ cm in the normal lung extracts and 23.1 dynes/cm in the airless lobes (Tables 1 and 2). A significantly lower stability index was calculated for re-expanded lobes which had been atelectatic for longer than 24 hours. Lobes atelectatic for seven hours are no less stable than normal lobes. There is no significant difference in maximal and minimal surface tension. However, lobes atelectatic for 18-96 hours all have significantly higher surface tensions and lower stability indices. The re-expanded lobes of the sarn~ lungs, however, have no significant decrease in surface activity through 24 hours of atelectasis, but definite abnormality thereafter (Fig 3 and 4). DISCUSSION

The question of the influence of alveolar collapse upon the integrity of pulmonar\' surfactant has received some attention. vine and Johnson'" investigated this phenomenon by using different methods of extraction of rabbit lung degassed after death. Their findings demonstrated the difficulty in extracting surfactant by the usual meth'ods from normal lung with collapsed alveoli. Further evaluation by the same authors of rabbit lungs collapsed in vivo indicated it surface tension abnonnalitv after 24 hours of collapse together with' an alteration of pressure-volume relationships. Abnormal pressure-volume determinations with saline and evaluation of deflation limb suggested that these changes were not primarily due to surfactant loss. PattIe" ha also indicated that the re-expanded atelectatic lung suffers no loss of surfactant, as • have Yeh and associates. lO The current study of atelectatic dog lungs adds further evidence that alveola~' collapse, by decreasing the alveolar surface in contact with saline, renders the extrac~ion of surfactant from minced lung specImens difficult. This is shown by the marked

Volume H. No.3

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SURFACE ACTIVITY OF LUNG EXTRACTS

March. 1968

TABLE 1-1-1EAN 1-1AXIMAL AND MI1';IMAL SURFACE TENSIONS A 'D STABILITY IXDlcl';S IN NORMAL LOBES AND THOSE ATELECTATIC AND RE-EXPANDED AFTER DIFFERENT TIM" !NT"RVALS

y Maximal (Dynes/em)

Duration A telec tasis (Hours) Normal

°

y Minimal (Dynes/em)

38.5±4.9

Atelectatic 7 24 48 7'J. 96 Re-expanded 7

43.0±2.0 41.8±2.1

N.S. p
5.7±1.9 23.7±4.4 14.7±5.3 22.S±2.5 24.1±1.3 2 I .6±4.3

,So p
1.59±O.04 0.69±0.17 1.03±0.25 0,67±0.O7 0.55±0.07 0.64±0.20

p
·H.2±3.7 43,7±S.8 41.9±7.0 44.6±2.8 42,9±3.0 42.8::!:2.:5

N.S. N.S. N.S, p
4.5±O.9 11.1 ±7.0 9,7±8.0 13.9±8.0 17.3±6.4 18.0::!:10.4

p
1,60::!:0.O4 1.21±0.44 1.32::!:O.37 I.09::!:O.44 0.88::!:O,30 0.89±0.44

p
'~S.3±4.2

18

2-\48

72

96

1.44±0.16

7.0±2.l

41.6±5.9 48.7±2.6 44.8±3,4

18

S

(Dynes/em)

N.S.=Not significant.

disparity in surface forces between the collapsed and fully re-inflated lobes.

There was a difference in surface activity of extracts, however, in lungs atelectatic

ATELECTASIS WITH RE-EXPANSION • ATELECTATIC oRE-EXPANDED

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50

60

70

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90

100

TIME (HRS.)

FIGURE 3: Maximal and minimal surfacc tensions of extracts of atclectatic collapsed and re-cxpandcd lobes plotted against time interval following ligation {Jf bronchus. Means of 37 normal dog lungs arc shown with 1 and 2 standard dcviations.

260

Dist3StS of the ChtSI

SUTNICK, SOLOFF AND SETHI ATELECTASIS WITH RE-EXPANSION • ATELECTATIC oRE-EXPANDED

2.0 1.8

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x

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- - - - - - - - - - - - - - - - - - - - - - - - - - - I~

1.2

1.0 0.8 0.6 0.4 0.2

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10

20

30

40

50

60

70

80

90

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TIME (HRS)

FICURE 4: Stability index of extracts of atelectatic collapsed and re-expanded lobes ploued against time interval following ligation of bronchus. Mean of 37 normal dog lungs is shovm with 1 and 2 standard deviations.

for longer periods. Re-expansion of lobes atelectatic for no longer than 24 hours demonstrated surface forces not significantly different from the normal, while those of longer duration were distinctly different. Avery and Chernick" have indicated that pressure-volume curves suggest an abnormality in surface tension as well. However, Levine and Johnson,' who used both saline and air filling for their pressure-volume curves, implicated a tissue abnormality rather than a surfactant abnormality as the cause of altered lung inflatability. Decreased surface activity was also noted in surface balance studies, however.

The present investigation cannot solve this problem, but careful histologic examination by light microscopy revealed no differences in the appearance of the alveoli of the normal lobes and those that were inflated in vitro. We would not expect then that some residual alveolar collapse would be responsible for inadequate surfactant extraction. The possibility of other mechanical changes, perhaps ultrastructural, cannot be excluded. Avery and Chernick's suggestion" that atelectasis interferes wit h the blood flow to the affected area, decreasing surfactant production because of poor perfusion, merits further evaluation as the cause.

TABLE 2-MEAN MINIMAL SURFACE TENSIONS AND STABILITY INDICES OF LOBES ATELECTATIC Up TO 24 HouRs COMPARED WITH THOSE ATELECTATIC OVER 24 HOURS

y Min (Dynes/em) <24 Hours >24 Hours

Normal Atelectatic Re-expanded

7.0±2.1 16.1±8.2 p
<24 Hours 1.03±0.39 1.34+0.39

Stability Index >24 Hours

1.44±0.16 p
p
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261

'URFACE ACTIVITY OF LUNG EXTRACTS

Certainly it has been adequately demon~ strated in the past that there is a marked reduction in blood flow through the atelectatic lung. n .'3 Finley and co-workers" h a V e provided evidence that this influences surfactant production by showing that minimal surface tension becomes eIe vat e d within two or three hours after pulmonary artery occlusion, even before atelectasis can be demonstrated. Following bronchial ligation, however, atelectasis occurs promptly, but there is a considerable lag in loss of surface activity. This implies a drop in surfactant production because of decreased pulmonary arterial flow secondary to atelectasis. The microscopic slides were carefully reviewed to evaluate the presence of vasoconstriction or vasodilatation. Although this is largely a subjective evaluation, it was our impression that there was no significant difference between those lobes atelectatic for shorter or longer periods. This does not rule out hypoperfusion as the etiology of altered surface activity, however. Loss of surfactant secondary to atelectasis might be responsible for the mysterious pulmonary edema seen by Ortner" and Foucart'• following thoracentesis for m assi ve pleural effusion and the re-coIlapse of the lung following treatment of spontaneous pneumothorax. This phenomenon has been reproduced with artificial pneumothorax in rabbits,17 and because no bronchial obstruction was found, an intraalveolar etiology was proposed. It was reversible in five to seven days. Certainly, long-term collapse has been shown to cause a decrease in compliance, and anatomic changes have been demon, strated in such lungs. lB • • The earliest of these abnormalities is thickening of interalveolar septa, and later changes involve bronchi and pleura. The decreased compliance may be entirely on the basis of tissue changes, although surfactant may also be involved. The evidence presented that experimental obstructive atelectasis will cause a deficiency in detectable surfactant activity even

in fully re-expanded lobes suggests that atelecta. is itself exelts an influence upon surfactant integrity or extractability. That this occurs primarily in lobes collapsed for 24 hours or more, as noted in the work of others, suggests that one day may be the rate of surfactant turnover in normal dog lungs. Previous observations? of diminished surface activity in airless dog lungs, atelectatic for much shorter periods, were probabl) due to decreased extraction of surfactant in these specimens because of poor alveolarsaline contact. The importance of re-expanding collapsed lung specimens before surfactant extraction is stressed. SUMMARY

Atelectasis has been produced in 37 dogs by bronchial ligation for periods of time up to 96 hours. At necropsy, one lobe was fully re-expanded with air, and another left collapsed. Extracts of those lobes which remained atelectatic up to 24 hours demonstrated definite abnormalities in surface ac~ ti\ ity, while extracts of the corresponding re-expanded lobes were essentially normal. With more prolonged atelectasis, surface activity was abnormal in all specimens, but more so in those lobes not re-expanded. These findings emphasize the necessity of complete expansion of lung specimens before ~urfactant extraction. They lend sup~ port to the concept that an alteration of surfactant production occurs following atelectasis. and may be . econdar\" to a local perfusion. . decrease in alveola~ ACK:>IOWLEDGMENTS: The authors wish to Baker and Dr. Andrew .J. Donthank Dr. Fr~derick 1l~l1y for the~r careful review of the histologic shdes and Shuley Bcrk for her technical assistance.

RESU~IEN

Treinta y siete perros fueron sometidos a la atclecta~ia pulmonar mediante ligadura bronquial pOl' pCl'lodos hasta de 96 horas. En la autopsia se procedio a la rc-expansion de un lobulo pOl' insuflacion de aire dejando a 011'0 colapsado. Los cxtractos de lobulos alelectasiados durante 24 horas mostraron anormalidadcs bicn definidas en los fenomcnos de superficic, mientras que los de lobulos re-expandidos fueron normales. Con atelectasias mas prolongadas la actividad de superficic era anonnal ell lodos, pero cn mayor grado en los lobulos no rc-cxpandidos. Estas comproba-

SUTNICK, SOLOFF A D SETHI

ciones ponen de relieve la necesidad de re-expander completamcme los pulmones antes de la extracci6n de surfaclante. Asi mismo refucrzan cI conccpto de que Ia elaboraci6n de surfactante se altera durante la atelectasia, posiblemente dcbido a una disminucion local de la perfusion alveolar. ZUS"M~!ENFASSUNG

Atelektase wurde bei 37 Hunden durch Bronchusligierung £iir Zeiten bis zu 96 Stunden el'zeugl. Bei der Sektion wurde ein Lappen wieder vollstiindig mit Luft gefullt, wlihrend ein anderer kollabiert blieb. Extrakte aus denjenigen Lappen, die bis zu 24 Stunden atelektatisch blieben, zeigten eindcutige Veranderungen del' Oberflachenaktivitat, wahrend Extrakte aus den korrespondierenden wiederausgedehnten Lappen vollig normal sind. Mit steigender Dauer del' Atclektasen war die Oberflachenaktivitlit in allen Untersuchngsproben verandert, jedoch stlirker in den nicht wiederausgedehnten Lappen. Diese Ergebnisse unterstl'cichen die Notwendigkeit einer vollstandigen Ausdehnung von Lungenproben VOl' del' Extraktion oberflachenaktiver Stoffe. Sie unterstutzen die Auffassung, da/3 eine Anderung del' Produktion oberflachenaktiver Stoffe nach Ateleklase eintritt, die moglicherweise Foige einer 10kalen Abnahme del' alveolaren Durchstromung ist, REFERENCES FINLEY, T. N., SWENSON, E. W., CLEMENTS, ]. A., GARDNER, R. E., WRIGHT, R. R. AND SEVERINGHAUS, J. W.: Changes in mechanical properties, appearance and surface activity of extracts of one lung following occlusion of its pulmonary artery in the dog, The Physiologist, :~: 56, 1960. 2 SUTNICK, A. 1. AND SOLOFF, L. A.: Atelectasis wi th pneumonia: a pathophysiologic stu d y , Ann. Int. Med., 60:39, 1964. 3 AVERY, M. E. AND SAID, S.: Surface phenomena in lungs in health and disease, Medicine, 44: 503, 1965. 4 CLEMENTS, J. A.: Surface phenomena in relation to pulmonary function, The Physiologist, 5: 11, 1962. 5 LEVINE, B. E. AND JOHNSON, R. P.: The surface activity of saline extracts from inflated and degassed normal lungs, The Physiologist, 6: 225, 1963.

Di.e.ses oi the Ches'

6 LEVINE, B. E. AND JOHNSON, R. P.: Surface activity of saline extracts from infla:ted and degassed normal lungs, ]. Appl. PhyslOl., 19:333, 1964. A. 1. AND SOLOFF, L. A.: Surface 7 SUT~ICK, tension reducing activity in the normal and atelectatic human lung, Amer. ]. M ed., 35: 31, 1963. 8 CLEMENTS, J. A., H\;STEAD, R. F., JOHl'OSO:-l, R. P. AND GRJDETZ, 1.: Pulmonary surface sion and alveolar stability, j. Appl. Physlo/., 16:444, 1961. .. 9 PATTLE, R. E.: Properties, function and ortgm of the alveolar lining layer, Proc. Roy. Soc., B, 1'~8:217, 1958. H 10 YEH, T. J., ELLISON, L. T., MANNINO, ., HAMLIN, J. T., III AND ELLISON, R. G.: veolar surfactant in experimental atelectaSIs, Clin. Res., 13: 50, 1965. . 11 AVERY M. E. AND CHERNICK, V.: Alteratl.on of the' alveolar lining layer in living rabbits, j. Pediatric!, 63: 762, 1963. 12 MOORE, R. L.: The volume of !:>Iood flow per minute through the lungs followmg collapse of one lung by occlusion of its bronchus, Arch. Surg., 22: 225, 1931. . I 13 KEELY, J. L.· AND GIDSON, J. G.: Expenmenta atelectasis in dogs, Surgery, 11 :527, 1942. 14 FINLEY, T. N., TOOLEY, W. H., SWENSON, E. W., GARDNER, R. E. AND CLEMENTS,. J. A.: Pulmonary surface tension in e.xpertmental atelectasis, Amer. Rev. Resp. DIS., 89: 372, 1964. 15 ORTNER, N.: Zur Entstehung des a~uten Lu.ngenodems nach Thorakocentese, WIener Klmische Wochenschrift, 44: 1090, 1899. 16 FOUCART, E. J.: De la morte subite olf rapide apres fa Thoracocentese, A. Parent, Pans, 1875, 17 CARSON, R. J., CLASSEN, K. L., GOLLAN, F., GODDEL, W. G., JR., SHE RMAN, D. E. AND CHRISTENSEN, R. 0.: Pulmonary edema following the rapid re-expansion of a totall~ lapsed lung due to a pneumothorax: a chmcal and experimental study, Surg. FOTllm, 9: 367, 1958. 18 lll(N I'I£I_D, J. R., RATTENDORG, C., GAGO, 0., IGRO. S. L. AND AOAMS, W. E.: Pulmonary cOlllpliance following re-aeration of the chronically atelectatic lung, Surg. FOTllm, 13: 160, 1962. 19 CHWALIBOG, B.: The influence of partial collapse by pneumot~<:,rax on p~lmonary. parenchyma and bronchI 111 the rabbit, Gruzlzca, 31 : 131, 1963. For reprints please write: Dr. Sutnick, Institute for Cancer Research, 7701 Burholme Avenue, Philadelphia.

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