Pediatric Pulrnonology 13:203-208 (1992)

Original Articles -

Temperature Affects Mechanics and Stability During Initial Inflation-Deflation of Mature Fetal Lung Alan J. Mautone, PhD,’ Maria T. Antonio-Santiago, M D , ~Bella C. Clutario, MD,3 and Emile M. Scarpelli, MD, PhD4 Summary. Volumepressure (VP) curves of initial aeration of mature (0.94-0.97 term) rabbit fetuses were compared in three groups, respectively, at 37“C, with maximal inflation pressureof 25 cm HO , (P25); 22% P25; and 22”C, P30. Anesthetized fetuses were delivered through uterotomy; chest was opened; trachea of fetal pulmonary liquid (FPL)-filled lungs cannulated; and lungs inflated-deflatedin 5 cm H,O, 2 min steps under continuous microscopic observation. As distendingpressure was increased, FPL moved peripherally with airways inflation by free gas and with saccular recruitment by free gas and bubbles. Saccular aeration continued during initial reduction of P from Pma. At end-deflation, air was retained in saccules virtually exclusively as bubbles. Airways inflation required less P at 37°C though airways volume (V) was the same at both temperatures. Opening P was lower, and saccular aeration was larger and more rapid at 37°C. The apparently higher distensibility at 37°C was most likely due to temperature effects on fluid dynamics rather than on tissue elasticity. Maximal V attained during early P reduction in all groups, was total lung capacity (TLC) at 37°C and < TLC at 22°C. Air retention at end-deflation, with films of near-zero surface tension, was greatest at 37°C and least at 22°C. P25. Lung stability, greater at 37°C than at 22°C was best discriminated when V at PO was taken into account. This study shows that all quasistatic parameters of lung function are enhanced at 37°C; it provides a new perspective on the relative ease with which the mature fetal lung effects initial aeration at 37°C; and it establishes the requirement that assessment of perinatal lung function should take temperature into account. Pediatr Pulmonol. 1992; 13:203-208. 0 1992 Wileyiiss. Inc.

Key words: Bubble formation; onset of breathing; volumepressure curves; surface tendon; lung temperature.

INTRODUCTION At birth, the potential airspaces of the fetus are liquidfilled (“fetal pulmonary liquid,” FPL), so that the first inflation of the lungs requires dispersion of air into the liquid milieu.’ Frequently, lung mechanics of this event are evaluated by controlled inflation-deflation of animal lungs in vitro. Whereas most studies have been carried out at room temperature, the more relevant evaluation at temperature approximating body temperature has been adopted by some relatively recently. l o * ’ I The conventional standard is the volume-pressure (VP) diagram, in which inflation and deflation curves are plotted during either continuous or interrupted (“quasistatic”) movement of air into and out of the lungs. The VP construction is used to assess physiologically significant parameters, including opening pressure, aeration and recruitment of saccules, the neonatal analogue of 0 1992 Wiley-Liss, Inc.

alveoli, lung distensibility, and stability. However, the influence of temperature on the first aeration of the lungs has not yet been documented quantitatively. From the Departments of Anesthesiology and Physiology, UMBNew Jersey Medical School, ,Department of Pediatrics, UMD-New Jersey Medical School, and Children’s Hospital of New Jersey,2 Newark, New Jersey; Blythedale Children’s Hospital, Valhalla, New York,3 and Perinatology Center of Cornell University Medical College, New York, Ncw Y ~ r k . ~

Received October 4, 1991; (revision) accepted for publication April 14, 1992. Address correspondence and reprint requests to Dr. A.J. Mautone, Departincnt of Anesthesiology, UMD-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103.

Dr. Mautonc is the recipient of a FIRST award from the NHLBl of the National Institutes of Health, HL-38303.


Mautone et al.

We first reported that increase of temperature from 22 to 37°C significantly improves the quasistatic mechanics of the lung. lo We have now extended these observations in a quantitative comparison of initial inflation-deflation of mature fetal lungs at the two temperatures. MATERIALS AND METHODS

Twenty-three fetuses (body weight, 40.8 k 1.2 g, mean f SEM) from 8 New Zealand White rabbits were studied between 29 and 30 days gestation (0.94-4.97 term). Does were anesthetized with sodium pentobarbital, 30 mg/kg iv. Each anesthetized fetus was delivered through a uterotomy, as the neck was ligated to prevent air entry into the lungs. The sternum was split and the chest wall pinned away from the lungs. A polyvinyl catheter was inserted into the trachea through an incision below the larynx. Entry of FPL into the catheter and microscopic inspection of the liquid-filled lungs confirmed airlessness prior to inflation. The heart was excised in some fetuses. For studies at 37 k 1”C, the fetus was placed inside a sealed plexiglas box. A water reservoir, 2 cm below the fetus, was warmed by copper tubing through which thermoregukdted water circulated. Air within the box was humidified from the internal reservoir. An external air blower was used to maintain a 7 x 7 cm “dry” plexiglas surface through which the lung was viewed. The tracheal catheter was connected to a 5-mL pipette on line with a 2.4-L cylinder. Using Tygon tubing, a continuous water column was established from the cylinder (outside the box) to the pipette (within the box) in which an air column was interspersed between the water column and FPL within the tracheal catheter and lungs. A length of the tubing was coiled within the box to facilitate temperature equilibration of both air and liquid. The water column formed a meniscus in the calibrated pipette from which air volume was monitored. Lung temperature was monitored continuously by thermistors placed between the posterior surface of the lung and the chest wall. Separate thermistors monitored air temperature at the pipette orifice as well as box temperature. For studies at room temperature, 22 t 1”C, the lung, the 2.4-L cylinder, the pipette, and connecting tubing were in contact with room air. The lungs were kept moist with normal saline. The methods used for lung inflation-deflation were described previously. l3 Lungs were inflated and deflated by changing the water level in the 2.4-L cylinder in 5 cm H,O steps. Pressure (P), which was held at each step for 2 min, was increased from atmospheric (PO), to a maximum (P,,,,) then returned to PO. (Pressure increases are designated by the P level followed by I, e.g., PIOI, and decreases by D, e.g., PIOD). Volume (V) was recorded from the pipette at each step. The entire preparation was

monitored for leaks throughout each experiment. The system, from Tygon tubing through intratracheal catheter, was checked for leaks under water while applying maximal P to that part of the system after the completion of each experiment, leaks from the lung parenchyma could be detected easily by continuous microscopic observation of the moist lung surface. All preparations in which leaks were detected from any site were excluded. Fetuses were entered randomly into the study groups. Nine were studied at 37°C (five with heart excised and four with heart intact). We selected 25 cm H20as P,, because pilot studies revealed that a P of 30 cm H20 produced leaks in about 35% of lungs at 37°C. Six fetuses (three with heart excised and three with heart intact) were studied at 22°C at a P,, 25 cm H,O and eight fetuses (four with heart excised and four with heart intact) were 30 cm H 2 0 . P25 at 22°C was chosen studied at 22°C P, for direct comparison with fetuses at 37”C, P25. P30 was chosen to increase the maximal volume (V,,,) of 22°C lungs, which at P25 was less than V,, of the 37°C lungs. Volumes were corrected for compression within the delivery system and standardized to 37”C, dry. From these data, mean k SEM was calculated for V at each inflation4eflation step; V was expressed as a function of fetal body weight (mL kg- I ) and plotted on P as VP diagrams. A two-way analysis of variance for unpaired data was used to determine statistical significance between groups, with P < 0.05 accepted as significant. Lungs were observed through a dissecting microscope (Carl Zeiss, Germany) and periodically photographed, At end-deflation the lung surface was incised either with a scalpel or by microdissection as previously described. l4 Bubbles delivered by these maneuvers into the air-equilibrated normal saline solution that bathed the lungs were observed for 30 min. Bubble diameter was assessed from an eyepiece graticule at -- 3 min and at 30 min after delivery from the lung. Bubbles were also transferred to deaerated normal saline solution to test bubble film permeability to air. The VP data were also used for the following calculations: AVIAP (mL * kg-’/cm H20),an expression related to lung distensibility or compliance, was determined for airways inflation, inflation to P,,,,, inflation to V,,,, and deflation at both PI OD and P5D. The “stability index” of and the “expansion Gruenwald,’”(2V5 + V 10)/(2VmaX), index” of Clements et al. ,I7 (V5-Vd..)/(Vma, - Vds),both of which assess lung stability during deflation, were also calculated. In these equations, V5 and V10 are volumes retained at P5D and PIOD, respectively. V,, in the “expansion index” is estimated anatomic dead space, which we took to be 2.2 m L *kg-’.’* We also calculated a “retention index,” (VO - vds)/(v,n,, - Vdh), for each group, which is the fraction of V,, retained in saccules at end-deflation .



Temperature Affects Inflation-Deflation of Mature Fetal Lungs


TABLE 1-Volumes (V), in mL . kg-' (Mean f SEM) During Initial Increase (I) and Decrease (D) of Distending Pressure (P) in Mature Rabbit Fetal Lungs at 37 and 22°C P at which V was measured

37°C (n = 9)

P51 PI01 P151 P201 P251

2.0 2 0.5 5.4 k 1.4 9.3 2 2.1;' 38.7 f 5.6h 94.6 ? 5.5'

P301 P25D P20D P15D PlOD P5D

NA NA 106.5 f 6.3 107.5 f 6.5d 102.1 ? 6.4 92.1 ? 6.3 52.3 2 6.3


22°C (n = 14) 1.6 f 0.5 3.4 f 0.6 4.9 f 0.7 7.9 f 0.9" 21.8 f 4.4b ( n = 6) 48.8 k 5.4' 62.6 f 3.3 62.9 f 3.9d 56.8 f 3.2 51.7 3.6 44.5 ? 4.5 16.4 f 4.0


( n = 8)

NA NA 29.6 f 6.7* 28.3 ? 6.0 25.0 2 5.5 20.4 f 4.4 3.7 ? 1.0

NA, not applicable. aAt end of airways inflation, no significant differcnce. bAt first step of saccular aeration, V 37°C > 22°C. 'After additional inflation step, V 37°C > 22°C. dAt V,,,,, V 37°C > 22°C P30 > 22°C P25. Note: At decreasing P steps, P20D through PO, V at 37°C > 22°C P30 > 22°C P25.

Relative VP Curves Mean V at each P step is given in Table 1 and plotted as VP curves in Figure I . Since there was no difference in V between each 22°C group form P51 through P251, the groups are combined in this P range. Relative V (mL kg-'), which describes the real V of inflatable units on a standard scale, is useful for direct comparisons among groups of V-related anatomic changes at each P step. We saw that only conducting airways were inflated as P was increased from PO to P15I (37°C) and from PO to P20I (22°C). Thus, airways were inflated to the same V at both temperatures, albeit airways at 22°C required higher P. Saccular recruitment began when P was increased above P15I (37°C) and P20I (22"), i.e., opening P was lower at 37°C. Recruitment was seen as sequential infla0 6 10 16 20 26 30 tion of clusters of saccules, with peripheral, subpleural clusters generally preceding more centrally located units Pressure (cm H,O) in a pattern of "axial filling" comparable to that reported for immature fetal lungs.I3 The air/FPL column entered Fig. 1. Relative VP diagrams of first aeration of mature rabbit terminal units both in the form of elongated (nonspherifetal lungs in which volume is related to body weight. Pressure cal) bubbles and as free gas. Whereas bubbles could be was changed in 2 min steps of 5 cm H,O. Arrows indicatepoints seen (Fig. 2), entry of free gas was deduced from saccular of maximum volume (VmaX)for each diagram. 0 3PC, P25; o enlargement where there was no apparent bubble move22"C, P25; A 22%, P30. Data from Table 1. ment. During the initial phase of saccular aeration, V at 37°C (P20I) was greater than V at 22°C (P251). Similarly, when P was increased an additional step, V at 37°C (P25I) was greater than at 22°C (P30I). RESULTS Volume increased as P was initially lowered from Results from fetuses with heart excised were not sig- P,,,. It increased further during the second P reduction nificantly different from those with heart intact in each step in both the 37°C and the 22°C P30I groups. V,, group. Data were, therefore, combined. therefore, did not coincide with P,,, (Table 1 and


Mautone et al.

Volume at PO was highest at 37°C intermediate in the 22"C, P301 group, and lowest in the 22"C, P251 group (Table 1). For each group, saccular V at PO was maintained by bubbles of air. Bubbles came from each surface incised by scalpel in all regions of the lung from hilum to periphery, while microincisions of subpleural saccules invariably yielded bubbles and, after bubbles were delivered, the saccules from which they came had become airless (Fig. 2). Bubbles, which became spherical when delivered into the air-equilibrated saline medium (Fig. 2), did not changes size (range, 50 to 200 p m diameter) for at least 30 min at both 22 and 37°C. In accordance with the criteria of Pattle,lS this indicates that bubble stability and surface tension near zero are the same at both temperatures. In contrast, bubbles delivered into deaerated saline solution decreased rapidly in size and disappeared within 5 min, indicating that the bubble films are air-permeable. I s Distensibility, Stability and Retention

Fig. 2. Light micrographs of terminal units from mature rabbit fetal lung at PO after first inflation-deflation cycle. P, was 30 cm H,O, 22°C. x150. (A) All aerated structures contain formed bubbles. Photograph taken immediately after subpleural microincision; black arrows indicate the length of a terminal unit (containing elongated bubbles) at the end of which is a rent (white arrow) in the saccular wall from which bubbles quickly entered the bathing medium. (B) Same area after several bubbles had moved out of the unit so that volume is reduced considerably. The black arrows here coincide with the same loci indicated by black arrows in A. The only air remaining in this unit is in bubbles that have not yet entered the bathing medium (e.g., white arrow). Bubbles establish infrastructural support and aeration of the units. One of the free bubbles, now spherical, is seen in the medium (top center).

Fig. I ) . While this phenomenon was visibly related to saccular distention, bubble formation and recruitment could not be excluded as contributory factors. All saccules appeared aerated at V,, , 37"C, whereas aeration was not to capacity in both 22°C groups, i.e., uninflated regions were visibly interspersed with clusters of inflated saccules.

At comparable points of saccular aeration (Table 2), AV/AP of the 37°C group was about twice that in the 22"C, P301 group and 4 to 5 times greater than in the 22"C, P251 group. The range of values for stability index among all groups, 1.33-1.11, falls within normal limits as reported by Gruenwald.'" The range of expansion index (0.92-0.78) was comparable among groups, and may be considered normal. " The retention index, or saccular volume retained at end-deflation (PO) as a function of saccular volume at end-inflation (V,,,,), for the 37°C group was = 2 times greater than for the 22°C. P30I group and = 6 times greater than for the 22"C, P251 group. DISCUSSION

Our study offers new insight into quasistatic mechanics of mature lungs during initial inflation-deflation from the fetal state at 37°C. 1. Quulitutively, volume history of the lungs is comparable at 37 and 22"C, i.e., airways are inflated to about the same V before opening P is reached; saccular aeration and recruitment are effected by peripheral movement of FPL with air entering saccules both as free gas and in the form of bubbles as P is increased; saccular aeration continues when P is initially lowered from P,,,,; and air is retained in saccules virtually exclusively as bubbles at end-deflation (PO). This sequence is in agreement with previously reported studies at 22°C. 2. Airwuys inflution requires less P at 37°C than at 22°C to produce comparable V displacement. Thus, the higher temperature facilitates airways clearance of FPL and reduces opening P. Assuming resting airways V as


Temperature Affects Inflation-Deflation of Mature Fetal Lungs


TABLE 2-Indices of Lung Distensibility (AWAP), Stability (Stability Index and Expansion Index), and Volume at End-Deflation (Retention Index) at 37 and 22°C ~

37°C (Pmax= P2SI)

AV/AP ( H,O) Airways inflation Inflation to , P Inflation to V,,, Deflation at PlOD Deflation at P5D Stability index (2V5 V10)/(2Vm,,) Expansion index (V5 - VdsMVma, - VdJ Retention index (VO - VdJ(Vrn~,, - VdJ






22°C (P,,,x = P251)

0.62 3.78 7.17 10.21 18.40 I .33

0.40 I .67 3.15 5. I7 8.90 1.12

0.40 0.87 I .48 2.50 4.08 1.11




0.5 I



V10, V5, and VO are V at PIOD, P5D, and PO,respectively; V,, is estimated anatomic dead space Ix;V,,, and P,,,, are maximal V and P, respectively.

about 2.2 mL * kg-',18 we may estimate that airways are distended about 3.5- to 4.2-fold in V and about 2-fold in diameter prior to opening P. This is consistent with studies of adult" and fetali3 airways and it is reasonable to conclude that airways distention proceeds to the same geometric end-point prior to saccular aeration at both temperatures. However, the process required less applied force and, thus, is facilitated at the higher temperature. This could be due to facilitated peripheral flow of FPL and/or temperature-related increase of airways distensibility at 37°C. 3. Saccular aeration to total lung capacity (TLC) requires relatively low P and is effected relatively rapidly at 37"C, with approximately 88% V,,, delivered as P is increased to P251, and the remainder as P is initially reduced from P,, to P15D. The conclusion that V,, at 37°C is TLC for this stage of development is supported by observations that 37°C lungs appear fully inflated by microscopic examination; that attempts to increase V by increasing P above P25I often resulted in tissue rupture; at 37°C is in the range of highest V and that the V,, recorded for this age group by Kotas and Avery*' at room temperature, but with higher distending P and chest wall intact. Thus, under conditions of our experiments, inflation of saccular units at 37°C occurs within the range of P15I-P251-P15D. While total aeration of saccules in vitro is effected in minutes at 37"C, it is reasonable that the same in vivo, where distending P in the range of our study is applied intermittently for short periods,21 requires substantially more time. In contrast, saccular aeration and recruitment at 22°C requires higher distending P (P20I-P30I-P20D) to achieve about 59% TLC. The study of Kotas and Avery'" indicates that P35 or higher would have been required. However, such higher P is not consistent with distending P recorded in viva.*'

The reason for the apparently higher distensibility at 37°C may be related to temperature effects on tissue distensibility and/or fluid dynamics. The finding in adult lungs that tissue distensibility is minimally altered between 21 and 51"C22argues against the former possibility, while our own unreported observations that FPL viscosity is lowered significantly with a change in temperature from 22 to 37°C support the latter possibility. 4.Neither stability index nor expansion index discriminated lungs at 37 and 22°C. In this P range (PIOD-PSD), saccules are aerated with both free gas and bubbles. Since intrasaccular bubbles maintain saccular aeration without need for distending P (following section), we may speculate that the saccular free air/liquid lining-where there are no bubble films-produces a retractive force that requires equivalent counterpressure to sustain V at both temperatures. We suggest that this force is due to surface tension and tissue retraction, which at PlOD and P5D is low. 13*23Thus, surface tension at the free air/liquid interface must be greater than zero. Additional study is needed to resolve this issue. 5. Analysis of intrasaccular bubble film stability at both temperatures shows that bubble films are gas-permeable and reduce surface tension to zero, in accordance with the criteria of PattleI5 and previous studies. I4 Comparison of lungs at 37 and 22°C suggests that bubble production is enhanced by increased temperature, while comparison of the 22°C groups indicates that bubble production is also enhanced by increased aeration. Whether higher temperature or increased aeration per se is the primary factor needs further investigation. The present study shows that saccular V is maintained without distending P by the structural support imparted by bubble films, since V at Po is maintained by bubbles


Mautone et al.

of air and saccules collapse when bubbles are removed. We also demonstrate that all quasistatic parameters of lung function are enhanced at 37"C,and provide a new perspective on the relative ease with which initial aeration is effected in the mature fetal lung. Thus, assessment of perinatal lung function should take temperature into account.

Body temperature enhances quasi-static mechanical function of mature rabbit lungs. Pediat Res 1986; 20:470A. I I . Yamada T, Ikegami M, Jobe AH. Effects of surfactant subfractions on preterm rabbit lung function. Pediatr Rcs 1990; 27592598. 12. Burri PH. Development and growth of the human lung. In: Fish-


REFERENCES 1. Scarpelli EM. lntrapulmonary foam at birth: an adaptaptional phenomenon. Pediatr Res 1978; 12: 1070-1076. 2. Agostoni E, Taglietti A, Agostoni FA, and Setnikar I. Mechanical aspects of the first breath. J Appl Physiol 1958; 13:344-348. 3. Brumley GW, Chernick V, Hodson WA, Normand C, Fenner A,


and Avery ME. Correlations of mechanical stability, morphology, pulmonary surfactant and phospholipid content in the developing lamb lung. J Clin Invest 1967; 46:863-873. 4. ElKady T, Jobe A. Corticosteroids and surfactant increase lung volumes and decrease rupture pressure of preterm rabbit lungs. J Appl Physiol 1987; 63:161&1621. 5. Faridy EE. Air opening pressure in fetal lungs. Resp Physiol


1987; 68 :293-300. 6. Humphreys PW, Strang LB. Effects of gestation and prenatal asphyxia on pulmonary surface properties of the foetal rabbit. J Physiol 1967; 192:53-62. 7. Mitzner W, Johnston JWE, Scott T, London WT, Palmer AE.

Effect of betamethasone on pressure-volume relationship of fetal rhesus monkey lung. J Appl Physiol 1979; 47:377-382. 8. Scarpelli EM, Cataletto M, Clutario BC. The role of surfactants in the immediate neonatal period. J Perinatol 1986; 6:203-210. 9. Taeusch HW Jr., Wyszogrodski I, Wang NS. Avery ME. Pulmonary pressure-volume relationship in premature fetal and newborn rabbits. J Appl Physiol 1974; 37:809-813. 10. Antonio-Santiago MT, Mathew R, Clutario BC, Scarpelli EM.



man AP, Fisher AB, Geiger SR, eds. Handbook of Physiology, Section 3. The Respiratory System, Vol. 1. Bethesda, MD. American Physiological Society 1985. Scarpelli EM, Kumar A, Doyle C, Clutario BC. Functional anatomy and volume-pressure characteristics of immature lungs. Resp Physiol I98 I; 45:25-41. Scarpelli EM. Clutario BC, Mautone AJ, Baum J . lntrasaccular bubbles of near-zero surface tension stabilize neonatal lungs. Pflugers Arch 1984: 401:287-292. Pattle RE. Properties, function and origin of the alveolar lining layer. Proc Royal S o c London 1958; B148:217-240. Gruenwald P. A numerical index of the stability of lung expansion. J Appl Physiol 1963; 18:665467. Clements JA, Husted RF, Johnson RP and Gribetz 1. Pulmonary surface tension and alveolar stability. J Appl Physiol 1961: I6:444450.

Rahn H, eds. Handbook of Physiology, Section 3, Respiration, Vol. 1. Washington, D.C.. American Physiological Society, 1964. Scarpelli EM, Real FJP, Rudolph AM. Tracheal motion during eupnea. J Appl Physiol 1965; 20:473479. Kotas RV, Avery ME. Accelerated appearance of pulmonary surfactant in the fetal rabbit. J Appl Physiol 1971; 30:358-362. Saunders RA, Milner AD. Pulmonary pressure/volume relationships during the last phase of delivery and the first postnatal breaths in human subjects. J Pediatr 1978; 93:667-673. Inoue H, lnoue C, Hildebrandt J. Temperature effects on lung mechanics in air- and liquid-filled rabbit lungs. J Appl Physiol

18. Bouhuys A. Respiratory dead space. In: Fenn WO,

19. 20. 21.


1982; 531567-575. 23. Scarpelli EM. The lung, tracheal fluid, and lipid metabolism of the fetus. Pediatrics 1967; 40:951-961.

Temperature affects mechanics and stability during initial inflation-deflation of mature fetal lung.

Volume-pressure (VP) curves of initial aeration of mature (0.94-0.97 term) rabbit fetuses were compared in three groups, respectively, at 37 degrees C...
525KB Sizes 0 Downloads 0 Views