THE INFLATIONARY FORCEPRODUCEDBY PULMONARYVASCULARDISTENTION IN EXCISED LUNGS. THE POSSIBLE RELATION

OF THIS FORCETO THAT NEEDEDTO INFLATETHE LUNGSAT BIRTH

By MARYELLEN AVERY,* N. ROBERTFRANKAND IRWIN GRIBETZ t

(From the Department of Physiology, Harvard School of Public Health, Department ofPediatrics, Harvard Medical School, and Boston Lying-In Hospital,

Boston, Mass.)

(Submitted for publication August 18, 1958; accepted October 31, 1958)

Among the forces that may contribute to thefirst inflation of the lungs after birth, those de-veloped through changes in the pulmonary circu-latory system have been assigned primary impor-tance by Jaykka (1), Bonham Carter (2) andAdams, Karlberg and Lind (3). Possibly the ear-liest basis for this view was provided during thelast century by von Basch (4) who showed in ani-mals and also in experimental models that pulmo-nary vascular congestion, presumably at the capil-lary level, tends to increase the volume of gas inthe lungs. Recently, it was demonstrated in ex-cised cats' lungs that the expansile force resultingfrom vascular congestion is greatest when the lungsare collapsed (5). Together, these findings mightbe taken as evidence underscoring the role of vas-cular distention in expanding the lungs at birth.Before this view is accepted, however, two factsmust be established. One is that the increase inpulmonary blood flow precedes the first breath.This is contrary to the findings of Dawes in fetallambs that pulmonary blood flow is increased onlyafter inflation has occurred (6). The other is thatthe force developed through vascular filling or con-gestion is sufficiently large to cause air to enter thelung. The object of this study was to test this sec-ond possibility. Experiments were carried outto compare the distending forces produced by awide range of pulmonary vascular pressures withthe force required to inflate the lungs. Measure-ments were made on excised lungs of adult catsand dogs that were freed of gas to simulate theircondition at birth, and on lungs of puppies andnewborn infants that had not breathed.

* This work was supported in part by a Special Trainee-ship (BT-259) from the National Institute of Neurologi-cal Diseases and Blindness, National Institutes of Health.

t Research Fellow, National Foundation for InfantileParalysis, Inc.

METHODS

Excised lungs from four adult cats, one adult dog, fivefetal puppies and two stillborn infants were used. Theanimal lungs were made gas-free by a modification of thetechnique of Coryllos and Birnbaum (7). The cats wereanesthetized with 30 mg. per Kg. of pentobarbital (Nem-butal®) 1 injected intraperitoneally; the dogs were giventhe same dose intravenously. Ninety-nine plus per centof oxygen was used to ventilate the lungs through atracheal cannula for about 15 minutes, after which thechest was opened and the trachea was occluded to permitabsorption of the gas by the pulmonary circulation.Early in the period of gas-freeing 375 mg. of mepe-sulfate2 (an anticoagulant), diluted with saline, was in-jected into the left ventricle. The heart and lungs wereexcised together. Cannulae were inserted into the pul-monary artery and left atrium; a clamp was put acrossthe myocardium just below the mitral valve in order toclose the pulmonary vascular system. Each cannula wasconnected to a dispensing burette and pressure trans-ducer. To minimize the formation of pulmonary edema, 6per cent dextran in Ringer's lactate solution was used tofill the blood vessels. Trypan blue was added to thesolution to help detect leaks. A tracheal cannula was in-

I'.'I.... ..

I ....

PLUNG UPLA

PPA

FIG. 1. SCHEMATIC DIAGRAMOF BURETTE, CANNULAETO PULMONARYARTERY AND LEFT ATRIUM, AND INTRA-

TRACHEALCANNULAT indicates pressure transducer. Cannulae to pulmonary

artery and left atrium are represented by black spot be-

tween the lungs.

1 Abbott Laboratories.2 Hoffmann-La Roche, Inc.

456

EFFECTS OF VASCULARDISTENTION ON LUNG INFLATION

FIG. 2. DIAGRAM OF APPARATUSFOR MEASURINGPRES-SURE-VOLUMECHARACTERISTICS OF EXCISED LOBES $

A correction for compression of gas within the bottle A - 3 '> 'was made by clamping the trachea, elevating the burette, aand subtracting the volume change recorded from the final 470Dreadings with the lungs in the system. '-i 4

_4

serted close to the carina and connected to an inductance amanometer. The gas volume of the cannula and tubing -was kept small, ranging up to approximately 5 ml. Theapparatus for this part of the experiment is shown sche- v A-;. Gmatically in Figure 1.

By varying the height of the dispensing burette lead- Iing to the vascular system, pressure could be raised to any ' 'level in the pulmonary artery and left atrium. Vascular W,congestion was maintained approximately 15 seconds, ^ . -A i+.,

_ A FIG. 4. SECTION OF LEFT LUNG FROM SAME INFANTSif,#B,JJ;*$FE~t~tVi id <lP 9 AS IN FIGURE 3, AFTER INFLATION WITH AIR BY POSITIVE

hii

39 ;6'Tall, O--v s zPRESSUREAPPLIED TO TRACHEAX 100. Hematoxylin and eosin.

Otto unless otherwise stated in the figure legends. Gas wasA -J ! %X$0Q. . sa;notallowed to enter the lungs during these periods of

vascular congestion so that changes only in airway pres-sure were possible. All pressures were recorded simult aneously on a direct-writing oscillograph.

. 4 One human left lung with a weight of 18 Gm. was~~I~~~~ '~ studied four hours after death. It was obtained from a

_AdAdeA.,. i. i, ,1,500 Gm. anencephalic monster that made no respira-tory movements but did have a pulsating heart for 40minutes after delivery. The other human lung, from an

,'<Adt ' 1,880 Gm. anencephalic monster, was studied 7.5 hoursv;ssg5:r sa after stillbirth. The experimental procedure was the

k\ \ 5

same as that outlined for the animals except that no anti-oi coagulant was used, and the ductus arteriosus was tieds.i;as;Ad;g~g~g off to produce a closed pulmonary vascular system.

- ~ ~ ~ Histologic sections of the lungs from the 1,880 Gm. in-~~T~~~h~~4~ ~ ~ '~~~~ fant are shown in Figures 3 and 4.~~~~~/~~~~~~~~The fetal puppies weighed from 140 to 240 Gm. and

-t~ t ,^I n 4hip were obtained from two pregnant dogs estimated to be5 t . near term. One of the dogs was sacrificed by injecting

nail<}AddAid -> ^ ! Nembutal® intravenously, after which eight puppies were

delivered as quickly as possible by cesarean section.FIG. 3. SECTION OF RIGHT LUNG FROM 1,880 GM. Mepesulfate was injected into the fetal hearts which

;TILLBORN ANENCEPHALIC MONSTERAFTER VASCULAR were beating at the time of delivery. The puppies didNGORGEMENTAND FORMATION OF PULMONARYEDEMA not breathe during the period of direct observation. TheX 100. Hematoxylin and eosin. other dog was anesthetized with NembutalS and the

457

MARYELLEN AVERY, N. ROBERTFRANK, AND IRWIN GRIBETZ

lungs ventilated with 99 plus per cent oxygen througha tracheal cannula. An attempt was made to preventintrauterine respiratory movements by injecting intra-venously 0.3 Gm. morphine sulfate to sacrifice the ani-mal. Before the puppies were removed from the uterus,the fetal tracheae were clamped; after delivery, the heartand lungs were excised and prepared in the manner al-ready described; in addition, care was taken to occludethe foramen ovale with a clamp placed across the leftatrium. Because of the extreme fragility of the cardio-vascular tissue, leaks caused by damage to the tissuecould not be avoided; as a consequence it was not pos-sible to measure vascular volume, although vascular pres-sure was recorded accurately.

The preparation used to measure pressure-volume char-acteristics of excised lungs previously made gas-free isshown schematically in Figure 2. The lungs were ob-tained from five cats, 2 to 3.5 Kg. in weight, and five dogs,14 to 18 Kg. in weight. Elevation of the dispensing bu-rette shown on the right forced gas into the lungs. Thedifference between fluid levels in the burette and bottlerepresented the distending pressure for the lungs; thispressure was read from a water-filled manometer. Pres-sure was raised in steps of 2.5 cm. of water until a largeportion of the lungs was inflated; thereafter, steps of 5cm. of water were used until a peak level of 35 cm. ofwater (occasionally 30 cm. of water) was reached. The

-- ,-'++-A an AmAn+£;n c+avc m; C of no -lUilgs were permiteLLdU tL UaeadLe 1

ter. Two minutes were allowedcur before changes in volume wwas read directly from the buremade for compression of the gas.run, the volume of the lungs wament of fluid.

To determine whether the distegas first entered the lungs coulconfiguration of the lungs, thesemade on individual lobes of dogs'filled with saline. Two methodsdirectly through the bronchial treithe blood vessels by raising vascuwhich "edema" fluid entered the;of fluid added was roughly equa

CAT

RT LUNG {ECM. H O

60-

PLA 40

-.0-

60-PPA 402

20-

4-6-

120 ISEC.

FIG. 5. PRESSURE TRACINGSFRC

DOG 5 KG. 9R.U L 20.5G

CM. H20 6-40-

pLA 202...0-

....

/

60-40-

PPA 20-

0- ~ ..

+ 2-:

PAIRWAy _2- X4-6-

20 1SEC.

FIG. 6. PRESSURETRACINGS FROM GAS-FREE DOG LOBE

tional residual capacity of the lobe, assuming that thedistribution of the functional residual capacity among thelobes is proportional to their weight. Values for thefunctional residual capacity were based on the data ofMead and Collier (8).

RESULTS

Influence of acute pulmonary vascular congestionon airway pressure in gas-free excised lungs

in steps .JoIII. UI wa- The findings in adult cats' and dogs' lungs werefor equilibration to oc- similar.ere measured. Volume Typical tracings from two experimentstte after correction was are shown in Figures 5 and 6. The control pul-

Before and after each monary arterial pressure was set at about -4 cm.Ls measured by displace- of water; in the experiments shown, left atrial

nding pressure at which pressure during the control period was set at 8 to

Id be influenced by the 10 cm. of water which is within the normal rangemeasurements were also for the intact animal. In other experiments, con-lungs that were partially trol pressure in the left atrium was set at -4 cm.were used to instill fluid of water without any apparent difference in re-e, and indirectly throughilar pres re tolvs sults. When arterial pressure was raised acutelyAar pressure to levels at

t 0c.o ae rmrpesr ntelfair spaces. The amount to 20 cm. of water or more, pressure in the left1 to the estimated func- atrium rose to nearly the same level within 15

seconds. Filling of the vascular system, judged27 KG from the distribution of the blue perfusate, ap-ri1mATEn WT 10 G. peared to occur throughout the lungs.

The range of pulmonary vascular pressure wasfrom 20 to 80 cm. of water. Vascular volume andpressure showed a close correlation even thoughtime was not allowed for complete equilibration tooccur (Figure 7). At lower vascular pressures,almost all of the fluid was regained upon emptying

-.t-~~; the system. At vascular pressures of approxi-mately 60 cm. of water or more, the recovery offluid was incomplete, suggesting that edema or"leakage" had occurred.

)m GAS-FREE CAT LOBE In all experiments, the change in airway pres-

458

EFFECTS OF VASCULARDISTENTION ON LUNG INFLATION

5A AIRWAYPRESSURE4

CM. H20 3

DOG 15 KG. QRU.L. 20.5G.

e 5-

3-

2-

I * ' *0 20 40 60 0 10 20PULM. ART PRESSURE A PUL. VASC VOLUME-ML

CK 120 (CALCLATED)

FIG. 7. THE RELATIONSHIP BETWEEN PULMONARYVASCULARPRESSUREANDVOLUME, ANDAIRWAY (TRANS-PULMONARY)PRESSURE

sure followed within a few seconds the onset ofcongestion. The direction of change was towardsubatmospheric; that is, the force which developedacted in the direction of expanding the lungs. (Itwill be recalled from the description of the prepara-tion that gas was not permitted to enter the lungs.)The change in airway pressure correlated well withthe level of vascular pressure, ranging from about1 cm. of water at a vascular pressure of 20 cm. ofwater, up to nearly 5 cm. of water at vascularpressures of 50 to 70 cm. of water. The largestdistending pressure developed in any lung in thisseries was 5 cm. of water.

At lower degrees of congestion, airway pressureapproached equilibrium asymptotically and re-turned nearly to control level when congestion wasreversed. At the highest vascular pressure, theresponse was biphasic: Initially, as before, airwaypressure became subatmospheric, but it shortly re-versed its direction and became positive relative tothe atmosphere if the congestion was sustainedlong enough. This reversal appeared to corre-spond to formation of increasing amounts of

DOG15 KG 9

RUL 205GCM. 20 o-

60-PLA 40-Z

oz

6-40

PPA zo ...

PA1RWAY 2- S 2.#6- 4-

FIG. 8. THE EFFECT OF VASCULAR CONGESTION ONAIRWAY PRESSURE, SHOWINGTHE CHANGEIN SIGN INTHE AIRWAY DtJRING EDEMA FORMATION

edema. (The lungs were seen to increase sig-nificantly in volume.) It is noteworthy that, sub-sequent to this step, the response of airway pres-sure to lower levels of vascular pressure was sig-nificantly reduced (Figure 8).

In the human stillborn lung, pulmonary arterialpressure could not be transmitted to the left atrium.For this reason the procedure was reversed andleft atrial pressure was elevated in an attempt toopen the vascular system from the other direction.There was no retrograde transmission of pressureeither, until atrial pressure reached 80 cm. of wa-ter; at this point arterial pressure rose suddenlyto about 70 cm. of water. Associated with this ex-treme elevation of vascular pressure was a slightbiphasic change in airway pressure; that is, therewas a transient negative phase followed by a posi-tive one (Figure 9).

The results of experiments on fetal puppies wereless clear-cut, probably because of leakage out ofthe vascular system. The largest change inducedin airway pressure during filling of the blood ves-sels was less than 1 cm. of water.

To ensure that pressure changes measured withthe gas-filled recording system (tracheal cannulaand pressure tubing) were a valid reflection ofchanges occurring at the alveolar level, two ad-ditional procedures were done. One was to placea gas-free cat's lung in an air-tight, stoppered glassjar; the lung was supported by its tracheal can-nula leading through the stopper to a transducer;several sizes of cannulae and lengths of tubing

STILLBORN (ANEN'CEPHALICG d 5 KG.LT. LUNG 18.0 G.

CM. H20 a-PAIRWAY O-

- 2-

80-

PLA 60--_40-:

60- tC\.40-PPA 20-_ i KO---- -J

120 ISEC.

FIG. 9. PRESSURETRACINGS OBTAINED DURING ELE-VATION OF THE LEFT ATRIAL PRESSUREIN THE LUNGSOF A HUMANSTILLBORN

Note the very minimal changes in airway pressure ac-companying vascular distention.

459

4MARYELLEN AVERY, N. ROBERTFRANK, AND IRWIN GRIBETZ

were used. Suction was then applied around thelungs in steps of 1 cm. of water up to a total of-6 cm. of water, while the pressure response in-side the trachea was recorded. In general, about80 per cent of the applied negative pressures wastransmitted to the manometer; that is, a pressureof -5 cm. of water around the lungs was read as-4 cm. of water. The small variations of size ofthe cannulae and tubing used in the first experi-ments did not significantly affect this degree oftransmission.

The second procedure was to repeat the experi-ment entirely under fluid (saline). In this sys-tem, zero reference pressure for both the lungsand blood vessels was the level of the saline bath.The assumption was made that in the absenceof an air-fluid interface the changes in pressureat the alveolar level would be transmitted in totalto the manometer. It was found that the magni-tude of change in airway pressure induced bygraded elevations of vascular pressure were ap-proximately the same as those in the conventionalpreparation.

Volume-pressure characteristics of the gas-freeand partially fluid-filled lungs

Typical examples of static volume-pressure char-acteristics of the separate lobes of a dog's lung areshown in Figure 10. To facilitate comparison ofthe lobes, intrapulmonary volume was expressed inml. (gas, fluid, or both) per Gm. of tissue. In thispreparation the right upper lobe was completelycollapsed, the left lower lobe was partially filledwith saline through its major bronchus, and theright middle lobe was partially filled with salinethrough its vascular system.

During inflation of the totally collapsed rightupper lobe there was no appreciable increase in vol-ume until a pressure exceeding 20 cm. of water wasapplied to the airway. Judging from the appear-ance of the pleural surface, filling was complete at35 cm. of water. On the other hand, equivalentinflation of the two lobes that contained fluid oc-curred at pressures of only 12.5 and 15 cm. of wa-

ter. These lobes also appeared completely in-flated (there were no obviously collapsed unitson the pleural surface) at a distending pressureof 35 cm. of water. The volume-pressure relation-ships of these lungs are illustrated in Figure 10.

VOLUMEML/GMLUNG

10-

5

0- o 5 ~105 20 25 30PRESSURE- CM, H20

*RUL 165 GM.

X LLL 27 GM.A RML 10 GM.

FIG.10. PRESSURE-VOLUMECURVES OF EXCISED GAS-FREE DOGLUNGS

The inflation curves are on the right, deflation on theleft. * indicates gas- and fluid-free right upper lobe;X, partially filled with saline through bronchus; and A,partially filled with saline through the vascular system.

During deflation, the curves for all three lobeswere roughly similar except at zero airway pres-sure at which the volume was greater in the twolobes containing fluid; this occurrence of an in-creased volume at zero airway pressure in thepresence of "edema" is typical for excised lobes(9).

DISCUSSION

Fluid was allowed to flow into, but not through,the vascular system from either side; approxi-mately equal levels of pressure were therefore ap-plied to all parts of the vascular system. Webe-lieve that the effect of congestion on the elasticbehavior of the lungs is exerted largely at the levelof the small vessels: the capillaries, and possiblyvenules, lying apposed to alveolar structures. Inmeasurements during life the left atrial pressurewhich is generally accepted as a reliable index ofcapillary pressure should provide similar informa-tion about the degree of congestion.

In general, the changes produced in airwaypressure acting to distend the lungs of the adultdogs and cats were roughly proportional to the

460

EFFECTS OF VASCULARDISTENTION ON LUNGINFLATION4

levels of vascular pressure that were applied.When the vascular pressure was raised to 30 cm.of water, an inflationary force of only 1 to 2 cm. ofwater was developed; at a vascular pressure of 40cm. of water it rose to about 2.5 cm. of water; atlevels of 60 to 80 cm. of water, the change in air-way pressure never exceeded 5 cm. of water.Vascular pressures of 30 to 40 cm. of water haveparticular relevance, for when these levels arereached in the left atrium of living dogs, pul-monary edema forms (9). The higher vascularpressures in these experiments were induced totest the extreme situation, answering the questionof how much effect on airway pressure is possible.

The relative lack of response among the lungsof stillborn infants and fetal dogs may have beendue to technical shortcomings: clotted blood whichprevented the transmission of pressure through-out the vessels (stillborn infants' lungs) and vas-cular leaks (fetal dogs' lungs). On the otherhand, the presence of amniotic fluid or secretionsin the lungs would tend to minimize the expansileeffect of vascular congestion. That is, the dis-tending force produced by a given degree of con-gestion is greatest when the lungs are at minimumvolume, and becomes progressively smaller as thelungs expand (5). Evidence for this was foundin the experiments in which vascular pressure wasmaintained at high levels for periods up to sev-eral minutes. Airway pressure, which at the out-set fell to subatmospheric levels, gradually re-versed its direction and finally became positiverelative to atmospheric pressure (Figures 5, 6, 8,9). Concurrently, the volume of the lungs, judg-ing from their external appearance, increased sig-nificantly even though the trachea was occluded.This suggests that the increasing volume of thelungs did minimize the inflationary effect of vas-cular distention.

Fluid which may be present in the passages ofthe lungs prior to initial inflation by gas will haveother implications as well. The results of theseexperiments suggest that if there is fluid equalin amount to half of the estimated functional re-sidual capacity, inflation will proceed at lower dis-tending pressure than if the lungs are totally col-lapsed (Figure 10). This effect may be due tochanges in the configuration of the smaller unitsof the lung. Fluid, by enlarging the radii of curva-ture of the alveolar ducts, could facilitate expan-

sion in accordance with the LaPlace relationshipwhich states that the pressure in a tube is directlyproportional to tension and inversely proportionalto the radius of curvature (P = T/r). In the caseof air entering a liquid-filled tube, the pressurerequired would be determined by the surface ten-sion at the liquid-air interface, and the formulawould then be P = 2 surface tension/radius (10).

The results of these experiments do not sup-port the hypothesis developed by Jiykkii (1) andamplified by Bonham Carter (2) and Adams andco-workers (3), that the force developed by"capillary erection" at birth is a primary factorcausing the first inflation of the lungs. Jaykkd'sevidence for the hypothesis rested on a series ofhistological sections from lungs of stillborn in-fants and animals; vascular congestion had beenproduced following excision of the lungs. Hefound that the lungs were more evenly expandedif macrodex 3 was first injected through the pul-monary artery than if they were inflated by gasdirectly through the trachea. The conclusion wasdrawn that circulatory adjustments at birth areresponsible for normal inflation. An alternativeexplanation for his histological findings is thatmacrodex which was injected into the pulmonaryartery under a pressure of 80 mm. Hg entered thealveoli as edema fluid, while the particles of Indiaink used to stain the fluid remained in the capil-laries. In the process of fixing the tissue macro-dex may have been washed out, giving the illu-sion of alveoli inflated by gas. Figures 3 and 4illustrate the difficulty of distinguishing betweenlungs that are filled with fluid or inflated with gas.Figure 3 shows a section from one lung of an 1,880Gm. stillborn infant after edema was inducedthrough the vascular system with macrodex stainedwith trypan blue. The other lung, which was in-flated with gas through the trachea, is shown inFigure 4.

SUMMARY

The role that increased pulmonary blood flow atbirth may play in expanding the lungs was studiedby measuring the pressures developed in the lungsduring acute vascular congestion. Measurementswere made in excised lungs of four adult cats, oneadult dog, five fetal dogs, and two stillborn in-fants. When the lungs were at minimum volume,

3 Pharmacia Ltd., Sweden.

461

4MARYELLEN AVERY, N. ROBERTFRANK, AND IRWIN GRIBETZ

the effect of vascular distension was in the direc-tion of facilitating expansion but it was not ofsufficient magnitude to initiate it. When the lungswere partially distended by fluid, the expansile ef-fect of vascular congestion was negligible.

The presence of a volume of fluid in the lungs,however, while minimizing the effects of vascularcongestion, permitted expansion at a lower airwaypressure than that required for lungs at minimalvolume.

ACKNOWLEDGMENTS

We are grateful to Dr. James L. Whittenberger andDr. Clement Smith for their advice and encouragement,and to Dr. Kurt Benirschke and the staff of the Depart-ment of Pathology of Boston Lying-In Hospital and Dr.John Craig of the Department of Pathology, Children'sHospital, for permission to study human lungs and fortheir help with the interpretation of histological sections.

REFERENCES1. Jaykka, S. Capillary erection and lung expansion.

Acta paediat. (Uppsala) 1957, 46, Suppl. 112, 9.2. Bonham Carter, R. E. The architectural function of

pulmonary capillaries. Lancet 1957, 1, 1292.

3. Adams, F. H., Karlberg, P., and Lind, J. Possiblerole of capillary erection as a cause for lung ex-pansion in the newborn infant. Clin. Res. 1958,6, 111.

4. von Basch, S. S. K. Klinische und ExperimentelleStudien, vol. 1. Berlin, August Hirschwald, 1891.

5. Frank, N. R. Effect of acute pulmonary vascularcongestion on recoiling force of excised cat lungs.In preparation.

6. Dawes, G. S., Mott, J. C., Widdicombe, J. G., andWyatt, D. G. Changes in the lungs of the new-born lamb. J. Physiol. 1953, 121, 141.

7. Coryllos, P. N., and Birnbaum, G. L. Studies inpulmonary gas absorption in bronchial obstruction;two new methods for direct and indirect observa-tion. Amer. J. med. Sci. 1932, 183, 317.

8. Mead, J., and Collier, C. Respiratory mechanics inanesthetized dogs. In preparation.

9. Cook, C. D., Mead, J., Schreiner, G. L., Frank, N. R.,and Craig, J. M. Pulmonary mechanics during in-duced pulmonary edema in anesthetized dogs. J.appl. Physiol. In press.

10. Mead, J., Whittenberger, J. L., and Radford, E. P.,Jr. Surface tension as a factor in pulmonary vol-ume-pressure hysteresis. J. appl. Physiol. 1957,10, 191.

ANNOUNCEMENTSOF MEETINGS

The Fifty-First Annual Meeting of THE AMERICANSOCIETY FORCLINICAL INVESTIGATION will be held in Atlantic City, N. J., onMonday, May 4, 1959, at 9:00 A.M. at the Casino Theater on the Steel Pier.

The Sixteenth Annual Meeting of THE AMERICANFEDERATIONFOR CLINICAL RESEARCHwill be held in Atlantic City, N. J., at theCasino Theater on the Steel Pier on Sunday, May 3, 1959, at 9:00 A.M.

THE ASSOCIATION OF AMERICANPHYSICIANS will hold itsSeventy-Second Annual Meeting in Altantic City, N. J., at the Casino Theateron the Steel Pier on Tuesday, May 5, 1959, at 9:30 A.M. and in the VernonRoom, Chalfonte-Haddon Hall, on Wednesday, May 6, 1959, at 9:30 A.M.

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