Respiration
(1975) 24, 217-232;
Physiology
DYNAMIC
North-Holland
ALVEOLAR
STUDIED
Publishing
MECHANICS
Company.
Amsterdam
AS
BY VIDEOMICROSCOPY
BENEDICT D. T. DALY, GEORGE E. PARKS, CHARLES H. EDMONDS. C. WAYNE HIBBS and JOHN C. NORMAN The Cardiooascular Texas
Children’s
Surgicul Hospitals
Research
Lubvrarories of
and the Departmenr
the Texas Heart
of Chemistry,
Rice
Instirute,
University,
St. Luke’s Houston,
Episcopal Texas.
und
U.S.A.
Abslract.
The in oioo morphology of rat alveoli has been recorded and studied on videotape. The dynamic internal structure has been compared to that observed by histologic section from lung rapidly frozen with liquid nitrogen. The irreversible nature of local alveolar wall movement has been demonstrated in animals ventilated both with and without positive end-expiratory pressure (10 cm water) by parametrically comparing the distances between two sets of paired points measured sequentially across internal alveolar surfaces at intervals of 0.017 sec. Geometric hysteresis was signifcantly less in the animals ventilated with positive end-expiratory pressure. This hysteresis can be explained by irreversibility in alveolar surface area apart from any irreversible dependence on surface tension. Sharp reverses and rapid changes in geometric hysteresis suggest that the length-tension properties of elastic tissue within the alveolar wall and interface forces from adjacent alveoli are important determinants of local alveolar wall motion and pulmonary hysteresis. Alveolar dimensions Alveolar wall Elastic tissue
Pulmonary hysteresis Pulmonary surfactant Rat
Morphologic and morphometric techniques have been refined and applied to explain the mechanics of respiration at the alveolar level. These approaches are appropriate since 90% of the lung volume is comprised of the alveoli and alveolar ducts (Weibel, 1963). Progressive increases and decreases in lung volume result in the pressurevolume hysteresis curve oflung( Mead et al., 1957). In normal lungs, three mechanisms have generally been invoked to explain this relationship. They are: the hysteretic properties of surfactant, the plastic behavior of lung tissue, and the irreversibility of recruitment and derecruitment of alveoli, i.e. the closing of alveoli in expiration does not exactly reverse the opening of alveoli in inspiration (Radford, 1964). The relative importance ofeach of these mechanisms in determining pulmonary hysteresis Accepted/or
publication
22 March
1975.
’ This work was supported by United States Public Health Service Contract No. 4173-196-4999-701 and a Grant from the American Heart Association. Texas Afiiliate. Inc.
218
B. D. T. DALY et al.
has not been resolved. The identification of a surface active material in lung by Pattle (1955) and the demonstration of its marked surface area-tension hysteresis by Clements et al. (1958) supports the contention that this material is responsible, at least in part, for compliance hysteresis in the lung. In addition, ultrastructural studies strongly support the presence on the alveolar surface of an extracellular lining layer which could represent surfactant (Weibel and Gil, 1968; Gil and Weibel, 1969/70; Gil, 1971), although the evidence is not incontrovertible (Brooks, 1971). However, the observation that compliance hysteresis is retained upon inflation and deflation with mercury (Pierce et al., 1961) suggests alternate or additional explanations since it is difficult to imagine that this heavy metal has affinity for even the hydrophobic end of the surfactant molecule. The contributions of the last two mechanisms have been considered minimal since hysteresis almost disappears when the lung is inflated with saline (Radford, 1957). However, Gil and Weibel (1972) demonstrated that sizeable numbers of collapsed alveoli were present in all lungs fixed at different points on their inflation and deflation cycles and that alveolar surface area changed in direct proportion to air volume. These observations support the thesis that alveolar recruitment does play a role in compliance hysteresis. They also noted that the mean curvature of alveoli changes in direct proportion to alveolar volume instead of alveolar volume to the two-thirds power, as would be expected for an expanding and contracting sphere. This could be explained by alveolar recruitment and derecruitment or by folding of the intra-alveolar septae within the surface lining layer. In a recent report, Hills (1971) demonstrated that the geometric hysteresis curves for excised human lungs correlated with their compliance hysteresis curves (P= 0.0003). He thus demonstrated an irreversible geometric relationship between area (assuming length is proportional to area) and volume, i.e. all dimensions which change upon inflation do not reverse exactly in magnitude or sequence upon deflation. He suggested this effect could be explained by treating surface tension as a manifestation of surface energy in a matrix such as lung. However, one aspect of geometric irreversibility is recruitment and derecruitment. His approach to compliance hysteresis treats the lung as a unit comprised of many alveoli and does not distinguish the possible effects of surfactant, the length-tension relationship of elastic tissue, or the micro-anatomy of lung tissue as contributing to surface energy at the alveolar level. In a recent report, Ardila et al. (1974) questioned Hill’s conclusions on theoretical and methodological grounds. They repeated similar experiments on freshly exsanguinated rabbit lungs supported by saline and demonstrated nearly equal inflation and deflation paths for orthogonally placed pleural markers while at the same time pressure-volume hysteresis was large. Nevertheless, some irregularities in sequencing and minor degrees of irreversibility were present, in their individual hysteresis curves. One drawback to these studies is the utilization of static morphologic and morphometric preparations in the investigation of a dynamic mechanical function of
DYNAMIC
ALVEOLAR
MECHANICS
219
lung i.e. hysteresis. Even momentary interruption of lung inflation and deflation can permit tissue to reach a stable pressure-volume configuration. Although techniques for the in uiuo visualization of dynamic alveolar morphology have existed for some time (Olkin and Joannides, 1930) relatively few studies have applied these techniques to the quantitative study of alveolar mechanics (Wagner, 1969; Moreci and Norman, 1972, 1973). In previous reports we have described an in uiuo video tape microscopy system which permits visualization of alveoli and alveolar sacs in rat lung (Daly and Norman, 1974; Daly et al., 1974). Their dynamic structure is recorded on video tape without interruption of respiration. Replay of the tapes with stepped slow motion, stop action and manual advance (one half frame at a time) permits morphometric analysis of dynamic alveolar function. The purpose of this report is to summarize our analyses of dynamic alveolar geometry during respiration and to relate these analyses to compliance hysteresis at the alveolar level. A preamble to this undertaking was a histologic correlation between what was observed in histologic section and what was observed in in Go microscopy to clarify the terms alveolus and alveolar sac as observed by the latter technique. Methods A.
LIGHT MICROSCOPY
The left pulmonary lobes of Sprague-Dawley rats weighing 250 g were inflated to a pressure of 20 cm HZ0 and immediately frozen with liquid nitrogen at - 196 “C. The lobes were then excised intact and fixed by freeze substitution (Staub and Storey, 1969). Sections from the anterior-inferior margin of these specimens, corresponding to the area of lung observed by in uiuo microscopy, were obtained by serial sectioning. Each section was 4 ~1thick. Maximum orthogonal alveolar diameters in two planes were determined for 31 alveoli and the mean alveolar diameter calculated. B.
IN WV0 MICROSCOPY TECHNIQUE
The anterior-inferior margin of the left lobe of 5 Sprague-Dawley rats was prepared for viewing with a videomicroscopy system by techniques described in previous reports (Daly and Norman? 1974; Daly et al., 1974). Briefly, ventilation via a tracheostomy was provided by a Harvard rodent respirator with a tidal volume of 4-6 ml at respiratory rate of 50 corresponding to the respiratory rate of the animals at rest. The airway pressure was continuously monitored at the outer end of the tracheostomy tube. For these studies, we utilized alveoli ventilated with positive pressure respiration both with an without 10 cm HZ0 positive end-expiratory pressure (PEEP). In a previous report PEEP was shown to increase both the total alveolar capacity or alveolar volume at peak inspiration, and alveolar functional residual capacity or alveolar volume at end expiration (Daly et al., 1974). In addition, PEEP was shown to increase the peak inspiratory pressure. Thus the effects of increased pressure and volume at total lung capacity and functional residual capacity on alveolar mechanics could be analyzed. A positive end-expiratory pressure
220
B. D.
T. DALY et al.
of 10 cm Hz0 was selected because it inflates the rat lung to nearly the top of its maximum pressure-volume (P-V) curve where atelectasis or recruitment and derecruitment should be eliminated. The videomicroscopy system consisted of a Wild-Heerbrug microscope with inverted epiobjective lenses, a Xenon arc light source, and a side-arm telescope connected in series with a television camera, image enhancer, time lapse video tape recorder (with stepped slow motion, stop action, and manual advance) and a video monitor. The inferior margin of the left lobe, stabilized by affixing pipe cleaners (approximately 0.5 cm apart) to its margins by medical adhesive, was approximated to a cover slip above the microscope objective. The traction and frictional resistance provided by the pipe cleaners prevented the left lobe from moving across the coverslip but did not inhibit 3-dimensional expansion and contraction of the lung enclosed within its margins. The area of lung studied was gently supported so that compression at the lung surface was minimized. Alveoli and alveolar sacs 1 to 1.5 mm below the pleural surface were visualized and their dynamic morphology recorded. c. ANALYSIS OF DYNAMIC
WALL
MOTION
For the investigation of the dynamics of alveolar wall motion, we applied the analysis of Hills (1971) for whole lung to individual alveoli. This analysis treats irreversibility or hysteresis as a function of surface energy, &. If the surface area of an alveolus is A, Es= yA, where y is the surface tension. Any loop in the pressurevolume curve could arise from an irreversibility in area apart from any irreversible dependence of surface tension on area, as assumed in attributing compliance hysteresis to surfactant alone. If geometric irreversibility can be demonstrated in an alveolus, then an irreversible relationship must exist between area and volume. It is possible the characteristics of geometric hysteresis curves could implicate the length-tension relationships of the alveolar wall and the stresses at alveolar interfaces as important determinants of hysteresis. The video recordings were replayed in slow motion and dynamic wall motion studied. Photographs of several alveoli and alveolar sacs were made from the video monitor at 0.033-see intervals to permit simultaneous comparisons between alveoli during different portions of the respiratory cycle. The alveolus from each rat which remained in the best focus throughout the respiratory cycle both with and without PEEP was selected for morphometric analysis. The technique utilized to stabilize the lung did not completely eliminate regional lung movement; it was, therefore, difficult to find more than a single alveolus in one field which was suitable for sequential morphometric analysis within a single breath. All alveoli, however, could be visually studied throughout a respiratory cycle by varying the focus. Two sets of paired points on the internal surfaces of the selected alveoli which could be followed accurately during respiration were identified. The distance or length (L) between these paired points (L, and L2) was measured directly from the video monitor. The videotapes were then advanced one frame at time throughout three breaths and
Fig. 1. A photomicrograph from a histologic section of rat lung frozen in uiuo and fixed by freeze substitution. The arrows point to folds in alveolar wails. x 480. Fig. 2. Each of these photographs was taken from images displayed on the television monitor, x 580. Schematic drawings of these images demonstrate the anatomic features of the alveoli. The solid areas in these schematic drawings represent alveolar walls; the stippled areas represent folds within the alveoli; and the clear areas depressions. (a) The arrow points to the wall of an alveolus. Its internal surface is comprised of many folds and depressions. These folds correspond to folds seen in histologic sections (fig. 1). The solid lines in the schematic drawing of this tigure connect paired points on the internal surface of the alveolus and represent typical L, and L, measurements.
(b) The same alveolus as in (a) 0.033 seconds later during uninterrupted respiration. Changes in the form and structure of the folds are evident. The solid lines in the schematic drawing demonstrate typical L 1 and L 2 measurements. The points of measurement correspond to identical points in the previous figure (2a).
(c) The portion of the mouth of an alveolus is seen directly below the O.
(d) The same alveolus as in (c) 0.033 seconds later during uninterrupted respiration. Changes in the form and structure of the folds are evident. The walls at the mouth of the alveolus have separated proportionately more than the remaining ·portions of the alveolar wall.
DYNAMIC
(e) Portions
of two alveolar
ALVEOLAR
MECHANICS
sacs are shown. The one on the left is comprised
3) and the one on the right of two alveoli (4 and 5).
223
of three alveoli (1, 2, and
224
B. D. T. DALY et ai.
the measurements repeated. Each measurement was repeated at 0.017~second intervals and an average of 65 measurements was made for each breath. These data were obtained from different alveoli by three different observers to eliminate possible bias in data acquisition. All measurements were entered on an IBM 35&155 computer via a time share terminal. L, and LZ measurements for each alveolusand each breath were then plotted separately by the computer as a function of time. A smooth curve was obtained by using a standard 5-point central difference smoothing technique. The smooth curves were then subtracted from the measured curves and the mean and standard deviation for the differences between all points on the measured and smooth curves were obtained for each breath. Tables were constructed by the computer for simultaneously obtained L, and L2 measurements for each breath utilizing every third point on the smooth curve. These time-paired L, and L2 points were then plotted. In order to identify artifacts introduced by the respirator, a rubber balloon was connected to the respirator, partially inflated and then further inflated and deflated by the respirator. Balloon wall motion was recorded on videotape. The changing dimensions of the balloon wall in a single plane were measured every 0.017 seconds and plotted as a function of time. Results
By light microscopy, the mean alveolar diameter from the exposed free edge of the left lobes of 250-g Sprague-Dawley rats inflated to 20 cm H,O pressure was 70.7 + 23.4 p (fig. 1). This measurement represents the average mean diameter since two orthogonal diameters from each of 31 alveoli were utilized in the computation. It is clear from these results that the structures illustrated in fig. 2ad represent alveoli and not alveolar sacs as we previously reported (Moreci and Norman, 1972, 1973; McNary et al., 1973; Daly et al., 1973; Daly er al., 1974). Mean alveolar diameter determined by video-microscopy techniques was 59.1+ 17.3 p. Alveolar sacs are illustrated in fig. 2e. These photographs were made directly from the television monitor and demonstrate not only the resolution obtained by these techniques but more importantly the variable internal structure of alveoli. Their internal surface is not smooth but rather punctuated by numerous folds and depressions which variably deform during respiration. The folds maintain their structural integrity from one breath to another. Figures 2a-b and 2cd show the changes in the internal structure of two alveoli which occurred 0.033 seconds apart. Figure 3 demonstrates the time changes in dimensions between one set of paired points measured across the internal surface of an alveolus during one breath. The smooth curve obtained by a central 5-point smoothing technique is also illustrated. Figure 4 shows similar curves obtained from a representative alveolus ventilated with 10 cm Hz0 PEEP. Several of the irregularities in these curves are due to respirator artifact. Figure 5 shows a time-dimensional curve obtained from a thin rubber balloon. The irregularities in this curve correspond to irregularities in the
225
Fig. 3. A time-dimensional curve showing the changes in the distances between one set of paired points across the internal surface of a normally ventilated alveolus throughout one breath. The points on the smooth curve selected for plotting hysteresis are shown as open circles and are connected by solid lines.
i TIME
110
&WW
Fig. 4. A timedimensional curve showing the changes in the distance between one set of paired points across the internal surface of an alveolus ventilated with positive end-expiratory pressure throughout one breath. The points on the smooth curve selected for plotting hysteresis are shown as open circles and are connected by solid lines.
TIME
&OMOI,
i
Fig. 5. A time-dimensional curve showing the changes in the distances across a rubber balloon in one plane as it is inflated and deflated by the respirator. The irregularities in the curve corresponded to
226
B. D. T. DALY
et ai.
above curves in time. While the changes in contour and irregularities of the curves from each set of paired points for each breath were similar, their relative contours changed from breath to breath. However, the changes were similar in each of the paired curves. L, and L, measurements for each breath were plotted parametrically. Frequently, the upslope and downslope of the resultant geometric hysteresis curves crossed each other at one or more points. In many instances, the reverses in these curves were abrupt. Although the curves for each breath in the same alveolus were different, the average slope of these curves was similar. Figure 6 illustrates a representative curve from a normally ventilated alveolus and fig. 7, one ventilated with PEEP. Several of these curves had multiple points of crossing at their base which represented points in time at the onset and termination of a breath. The upslope and downslope of the remaining portions of the curves had varying degrees of separation. This undoubtedly was affected by the actual location of the paired points IO-
sE 0
1 8b E
-I I-
& 1
6
6’ Fig. 6. A geometric rapid
reverses
hysteresis
curve for a normally
in this curve which are logically Maximum
7 LENCTN
point separation
Fig. 7. A geometric
hysteresis
b
ventilated
attributed
LEN&
curve for an alveolus
alveolus.
0.85 cm.
Note the sudden forces within
changes
the alveolar
and wall.
on the x axis and is I. I5 cm.
I- 2
ventilated
than that seen in fig. 6, and maximum
9
to mechanical
on this curve occurs
ecurve is smaller
I -cm
with positive
point
separation
end expiratory occurs
pressure.
The
on the y axis and
is
DYNAMIC
ALVEOLAR
227
MECHANICS
relative to the alveolar wall. The loops ofnormally ventilated alveoli were significantly longer than those for alveoli ventilated with PEEP with a single exception in the PEEP group. On reviewing these tapes, we discovered that one point from each set of paired points measured on the internal surface of this alveolar wall was adjacent to the mouth of an alveolus where it opened into an alveolar sac. Relatively greater wall motion was observed but not quantitated at similar points in other alveoli. Table 1 shows the maximum distance separating points on either the horizontal or vertical axis for each hysteresis curve in both the normally ventilated alveoli and those ventilated with positive end-expiratory pressure as well as the mean and standard deviation of the distance separating all points between the measured and smooth curves. True separation of any two points on these hysteresis curves demonstrated geometric irreversibility within the alveolar wall. Such separation was present for every curve in both groups. However, the separation was TABLE.1 Maximum
Alveoli ventilated
without
Alveolus-
Distance
breath
(cm**)
hysteresis point dikrential Alveoli ventilated
PEEP
Error S -.--
I-1
with PEEP
Alveolus-
Distance
breath
(cm) --.-
.--
PI
0.85
0.084 + 0.075
--1
Error
1.50
0.120*0.121
2
2.80
0.173kO.173
2
I.25
0.087 f 0.072
3
1.20
0.147f0.139
3
2.14
0.098 & 0.092 0.100+0.148
II -
I
0.90
0.148+0.113
- I
I .25
2
1.75
0.153kO.129
2
I.75
0.078 f 0.072
2
1.15
0.148+0.118
3
I.20
0.07 I + 0.060
PII’
111 - I
2.00
0.099 f 0.105
PI11 - 1
0.85
0.069 f 0.062
2
1.20
0.078 k 0.089
2
0.45
0.07 I f 0.082
3
2.00
0.077 k 0.084
3
0.70
0.067 f 0.065
IV - I
1.15
0.090 + 0.084
PIV - I
0.55
0.084 f 0.08 I
2
I.60
0.102 +_0.079
2
0.40
0.079 f 0.088
3
1.45
0.087 k 0.066
3
0.45
0.077 f 0.059
I
0.90
0.107+0.115
2
1.40
0.104kO.123
3
0.90
0.088 + 0.103
i
1.46
P < 0.05
SD
0.52
V’ -
1.01 0.60
* In rat V we were unable to follow points on the internal surfaces of an alveolus sequentially out repiration l
* All points were measured off the TV monitor 8 k+S.D.
for distance separating
in cm and plotted with the same units.
points on the smooth and measured curves for L, and L, of each
breath.
’
through-
with PEEP.
One point of each set of paired points of PI1 was at the mouth of an alveolus.
228
B. D. -I-. DALY et id.
significantly less (PC 0.05) in the PEEP group. Discussion In oiuo microscopy of the lung is not a new technique. Olkin and Joannides (1930), using a 6-volt light source and a microscope with 60 x magnification accurately described the living anatomy of alveoli. Since that time, other reports of in uiuo studies have appeared but these have mostly been devoted to the pulmonary microcirculation or to refinements in technique. DeAlva and Rainer (1963), Wagner and Filley ( 1965) and Sherman et al. (197 1) showed the in uiuo appearance of pulmonary alveoli but did not describe their dynamic morphology. The first in uiuo morphometric analysis of alveoli was performed by Wagner ( 1969). Although he reported quantitative measurements on alveolar diameters, he did not relate these to respiration. Moreci and Norman (1972, 1973) described the effects of positive pressure respiration on alvelar diameters of rat lung in inspiration and expiration and detailed their morphology. However, the terminal respiratory units visualized by incident light dark-field illumination were thought to represent alveolar sacs because of the many complete subdivisions observed within these terminal structures. In subsequent reports, the term “alveolar sac” was used in morphometric analyses of these structures (McNary et al., 1973; Daly et al., 1973; Daly et al., 1974). The measurements for “alveolar sacs”, however, correlate with measurements of rat alveoli obtained by others on fixed tissue (Tenney and Remmers, 1963; Macklin and Hartroft, 1943; Burri et al., 1974). The present investigation compares for the first time what is observed by histologic section and in uiuo microscopy. A rapid freezing technique using liquid nitrogen and freeze substitution was utilized to minimize tissue distortion in the histologic preparations. The animals used for these studies were prepared in the same manner as those utilized for in uiuo microscopy and comparable areas of lung were selected for study. Since the exposed lung was frozen and only the superficial alveoli were examined, liquid nitrogen was utilized in preference to liquid propane because of the hazards involved in the latter technique (Staub and Storey, 1969). In the histologic sections, alveolar diameters measured 70.7 k23.4 ,U and by in uiuo microscopy, 51.1 f. 17.3 p. These values are comparable. The lower figure obtained by in uiuo microscopy probably resulted from a lower mean inflation pressure. The lungs utilized for histologic study were inflated to 20 cm of H,O whereas the peak inflation pressure utilized in the in uiuo studies varied from 15 to 20 cm Hz0 (Daly et al., 1974). It is clear from these studies that the terminal respiratory units visualized in fig. 2acl represent alveoli and those in fig. 2e represent alveolar sacs. Our time lapse studies of alveolar wall motion have demonstrated the dynamic changes occurring within the alveolar wall with respiration. The folds observed in the walls of alveoli in histologic preparations would appear from our studies to represent the counterparts of the folds observed in uiuo. If this is true, these folds are not haphazard artifacts of tissue preparation but have both form and structure. The observation that wall motion at the mouths of alveoli is greater than wall
DYNAMIC
ALVEOLAR
MECHANICS
229
motion in other parts of alveoli indirectly supports the work of Macklin (1950) who expressed the view that the main increase in volume during inflation occurs in alveolar ducts. While we were unable to focus on alveolar ducts per se, we did observe relatively greater wall motion at the mouths of alveoli where two or three appeared together to form an alveolar sac. It is clear from our analyses that geometric irreversibility is one characteristic of alveolar wall motion. The sharp reverses and rapid changes in several of the observed geometric hysteresis curves are logically attributed,’ to mechanical wall properties and suggest that elastic forces within the alveolar wall or interface forces from adjacent alveoli are important in terms of local wall changes, Such forces were demonstrated in a mechanical lung model (Mead et al., 1970). Another aspect of dynamic irreversibility is the nonrepetitive wall movement observed in the timedimensional curves from breath to breath. Any error in the methods introduced by smoothing the curves is accounted for in table 1. The frequency with which the points for each curve were obtained and the acquisition of the points by three different observers eliminates any error introduced by observer bias. Although it is possible that the original points selected for measurement could have shifted within the alveolar wall as it thickened or stretched with respiration or changed its plane of focus slightly, these changes should have occurred equally for both dimensions measured. The time-dimensional changes recorded for a rubber balloon accounts for any respirator artifact. The report by Gil and Weibel (1972) supports these conclusions. Figure 11 in this report demonstrates irreversibility in a three-dimensional plot of alveolar surface area uerms both pressure and volume. Although Es= ?A, the gas in the alveolus contributes - PdV (inflation energy) to the total energy within the alveolar wall (P is the inflation pressure and V is the alveolar volume). The instantaneous wall energy within the alveolus can be expressed as dE = YdA- PdV. In a previous report (Daly et al., 1974) ventilation with a PEEP of 10 cm Hz0 was shown to inflate the rat lung to nearly the top of its P-V curves. Since A is proportional to volume to the 2/3 power, one could expect E, to decrease as V increased. This is in fact what was observed. At a PEEP. of 10 cm H20, geometric hysteresis was still observed but was less (PC 0.05) than that observed in the alveoli ventilated without PEEP. Although one aspect of irreversibility is recruitment and derecruitment, at this level of PEEP, alveolar recruitment and derecruitment should be minimally present if not absent. These studies demonstrate the dynamic nature of alveolar wall motion and the irreversibility of alveolar expansion and contraction. Thig can be explained by an irreversibility in alveolar area apart from any irreversible dependence on surface tension as assumed in attributing compliance hysteresis to surfactant alone. Recruitment and derecruitment of alveoli cannot completely explain this phenomenon. The characteristics of our alveolar geometric hysteresis curves both with and without PEEP suggest that the length-tension properties of elastic tissues, their distribution within the alveolar wall, and the interface forces from adjacent alveoli are important determinants of local alveolar wall motion and pulmonary hysteresis.
230
B. D. T. DALY et d.
Ardila, R., T. Horie and J. Hildebrandt (1974). Macroscopic isotropy of lung expansion. Respir. Physiol. 20: 105-I 15. Brooks, R. E. (1971). Ultrastructural evidence for a noncellular lining layer of lung alveoli. Arch. Intern. Med. 127: 426428. Burri, P. H., J. Daly and E. R. Weibel (1974). The postnatal growth of the rat lung. Anat. Rec. 178: 71 I-730.
Clements, J. A., E. S. Brown and R. P. Johnson (1958). Pulmonary surface tension and the mucus lining of the lungs: Some theoretical considerations. J. Appl. Physiol. 12: 262-268. Daly, B. D. T.. C. H. Edmonds and J. C. Norman (1973). In uioo alveolar morphometrics with positive end expiratory pressure. Surg. Forum XXIV: 217-219. Daly, B. D. T. and J. C. Norman (1974). Alveolar morphometrics 3: Preliminary results with in uiuo videomicroscopy. Chest 65: 67-68. Daly, B. D. T., D. A. Hughes and J. C. Norman (1974). Alveolar morphometrics: Effects of positive end expiratory pressure. Surgery 76: 624629. DeAlva, W. E. and W. G. Rainer (1963). A method of high speed in uiuo pulmonary microcinematography under physiologic conditions. Angiology 14: 160-164. Gil, J. and E. R. Weibe.1 (1969/‘1970). Improvements in demonstration of lining layer of lung alveoli by electron microscopy. Respir. Physiol. 8: 13-36. Gil, J. (1971). Ultrastructure of lung fixed under physiologically defined conditions. Arch. Inc. Med. 127: 896902. Gil, J. and E. R. Weibel(l972). Morphological study of pressure-volume hysteresis in rat lungs tixed by vascular perfusion. Respir. Phvsiol. IS: 190-213. Hills_ B. A. (1971). Geometric irreversibility and compliance hysteresis in the lung. Respir. Physiol. 13: SOdI. Macklin, C. C. and W. S. Hartroft (1943). Extramural Report to the Canadian Subcommittee on Physiological Aspects of Chemical Warfare. C.P. 35. Macklin, C. C. (1950). Alveoli of mammalian lung. Anatomical study with clinical correlation. Proc. Inst. Med. Chic. 18: 78-95. McNary. W. F., H. S. Berman, B. Daly and J. C. Norman (1973). The use of videotape to record and review data obtained by incident light dark-field microscopy of the living lung. Bib!. Anat. II : 4449. Mead, J., J. L. Whittenberger and E. P. Radford (1957). Surface tension as a factor in pulmonary volume-pressure hysteresis. J. Appl. Physiol. 10: 191-196. Mead, J., T. Takishima and D. Leith (1970). Stress distribution in lungs: A model of pulmonary elasticity. J. Appl. Physiol. 28: 596-607.
Moreci, A. P. and J. C. Norman (1972). Q uantitative and qualitative changes in morphology of alveolar sacs with positive pressure respiration. Proc. Sot. Expfl. Biol. Med. 141: 318-321. Moreci. A. P. and J. C. Norman (1973). Measurements of alveolar sac diameter by incident-light photomicrography. Ann. Thorac. Surg. 15: 179-186. Olkin, D. M. and M. Joannides (1930). Capillaroscopic appearance of the pulmonary alveoli in the living dog. Anat. Rec. 45: 121-127. Pattle, R. E. (1955). Properties, function and origin of the alveolar lining layer. Nature (London) 175: 1125. Pierce, J. A., J. B. Hocott and W. F. Helley (1961). Elastic properties and the geometry of the lungs. J. C/in. Inuesr. 40: 1515-1524. Radford, E. P. (1957). Recent studies of mechanical properties of mammalian lungs. In: Tissue Elasticity, edited by J. W. Remington. Washington, American Physiology Society, pp. 177-190. Radford, E. P. (1964). Static mechanical properties of mammalian lungs. In: XXII Intern. Congr. Sci., Vol. I, pp. 275-280. Sherman, H.. S. Klausner and W. A. Cook (1971). Incident dark-field illumination: A new method for microcirculatory study. Angialogy 22: 295-303.
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MECHANICS
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Staub, N. C. and W. F. Storey (1969). Relation between morphological and physiological events in lung studied by rapid freezing. J. Appl. Physiol. 23: 381-390;. Tenney, S. M. and J. E. Remmers (1963). Comparative quantitative morphology of the mammalian lung: diffusing area. Nature 197: 5456. Wagner, W. W. and G. F. Filley (1965). Microscopic observation of the lung in uioo. Vast. Dis. 2: 229-241. Wagner, W. W. (1969). Pulmonary microcirculatory observations in uioo under physiological conditions. J. Appl. Physiol. 26: 375-377.
Weibel, E. R. (1963). Morphometry of the Human Lung. Berlin, Springer. Weibel, E. R. and J. Gil (1968). Electron microscopic demonstration of an extracellular duplex lining layer of alveoli. Respir. Physiol. 4: 42-57.
(1975) 24, 217-232;
Physiology
DYNAMIC
North-Holland
ALVEOLAR
STUDIED
Publishing
MECHANICS
Company.
Amsterdam
AS
BY VIDEOMICROSCOPY
BENEDICT D. T. DALY, GEORGE E. PARKS, CHARLES H. EDMONDS. C. WAYNE HIBBS and JOHN C. NORMAN The Cardiooascular Texas
Children’s
Surgicul Hospitals
Research
Lubvrarories of
and the Departmenr
the Texas Heart
of Chemistry,
Rice
Instirute,
University,
St. Luke’s Houston,
Episcopal Texas.
und
U.S.A.
Abslract.
The in oioo morphology of rat alveoli has been recorded and studied on videotape. The dynamic internal structure has been compared to that observed by histologic section from lung rapidly frozen with liquid nitrogen. The irreversible nature of local alveolar wall movement has been demonstrated in animals ventilated both with and without positive end-expiratory pressure (10 cm water) by parametrically comparing the distances between two sets of paired points measured sequentially across internal alveolar surfaces at intervals of 0.017 sec. Geometric hysteresis was signifcantly less in the animals ventilated with positive end-expiratory pressure. This hysteresis can be explained by irreversibility in alveolar surface area apart from any irreversible dependence on surface tension. Sharp reverses and rapid changes in geometric hysteresis suggest that the length-tension properties of elastic tissue within the alveolar wall and interface forces from adjacent alveoli are important determinants of local alveolar wall motion and pulmonary hysteresis. Alveolar dimensions Alveolar wall Elastic tissue
Pulmonary hysteresis Pulmonary surfactant Rat
Morphologic and morphometric techniques have been refined and applied to explain the mechanics of respiration at the alveolar level. These approaches are appropriate since 90% of the lung volume is comprised of the alveoli and alveolar ducts (Weibel, 1963). Progressive increases and decreases in lung volume result in the pressurevolume hysteresis curve oflung( Mead et al., 1957). In normal lungs, three mechanisms have generally been invoked to explain this relationship. They are: the hysteretic properties of surfactant, the plastic behavior of lung tissue, and the irreversibility of recruitment and derecruitment of alveoli, i.e. the closing of alveoli in expiration does not exactly reverse the opening of alveoli in inspiration (Radford, 1964). The relative importance ofeach of these mechanisms in determining pulmonary hysteresis Accepted/or
publication
22 March
1975.
’ This work was supported by United States Public Health Service Contract No. 4173-196-4999-701 and a Grant from the American Heart Association. Texas Afiiliate. Inc.
218
B. D. T. DALY et al.
has not been resolved. The identification of a surface active material in lung by Pattle (1955) and the demonstration of its marked surface area-tension hysteresis by Clements et al. (1958) supports the contention that this material is responsible, at least in part, for compliance hysteresis in the lung. In addition, ultrastructural studies strongly support the presence on the alveolar surface of an extracellular lining layer which could represent surfactant (Weibel and Gil, 1968; Gil and Weibel, 1969/70; Gil, 1971), although the evidence is not incontrovertible (Brooks, 1971). However, the observation that compliance hysteresis is retained upon inflation and deflation with mercury (Pierce et al., 1961) suggests alternate or additional explanations since it is difficult to imagine that this heavy metal has affinity for even the hydrophobic end of the surfactant molecule. The contributions of the last two mechanisms have been considered minimal since hysteresis almost disappears when the lung is inflated with saline (Radford, 1957). However, Gil and Weibel (1972) demonstrated that sizeable numbers of collapsed alveoli were present in all lungs fixed at different points on their inflation and deflation cycles and that alveolar surface area changed in direct proportion to air volume. These observations support the thesis that alveolar recruitment does play a role in compliance hysteresis. They also noted that the mean curvature of alveoli changes in direct proportion to alveolar volume instead of alveolar volume to the two-thirds power, as would be expected for an expanding and contracting sphere. This could be explained by alveolar recruitment and derecruitment or by folding of the intra-alveolar septae within the surface lining layer. In a recent report, Hills (1971) demonstrated that the geometric hysteresis curves for excised human lungs correlated with their compliance hysteresis curves (P= 0.0003). He thus demonstrated an irreversible geometric relationship between area (assuming length is proportional to area) and volume, i.e. all dimensions which change upon inflation do not reverse exactly in magnitude or sequence upon deflation. He suggested this effect could be explained by treating surface tension as a manifestation of surface energy in a matrix such as lung. However, one aspect of geometric irreversibility is recruitment and derecruitment. His approach to compliance hysteresis treats the lung as a unit comprised of many alveoli and does not distinguish the possible effects of surfactant, the length-tension relationship of elastic tissue, or the micro-anatomy of lung tissue as contributing to surface energy at the alveolar level. In a recent report, Ardila et al. (1974) questioned Hill’s conclusions on theoretical and methodological grounds. They repeated similar experiments on freshly exsanguinated rabbit lungs supported by saline and demonstrated nearly equal inflation and deflation paths for orthogonally placed pleural markers while at the same time pressure-volume hysteresis was large. Nevertheless, some irregularities in sequencing and minor degrees of irreversibility were present, in their individual hysteresis curves. One drawback to these studies is the utilization of static morphologic and morphometric preparations in the investigation of a dynamic mechanical function of
DYNAMIC
ALVEOLAR
MECHANICS
219
lung i.e. hysteresis. Even momentary interruption of lung inflation and deflation can permit tissue to reach a stable pressure-volume configuration. Although techniques for the in uiuo visualization of dynamic alveolar morphology have existed for some time (Olkin and Joannides, 1930) relatively few studies have applied these techniques to the quantitative study of alveolar mechanics (Wagner, 1969; Moreci and Norman, 1972, 1973). In previous reports we have described an in uiuo video tape microscopy system which permits visualization of alveoli and alveolar sacs in rat lung (Daly and Norman, 1974; Daly et al., 1974). Their dynamic structure is recorded on video tape without interruption of respiration. Replay of the tapes with stepped slow motion, stop action and manual advance (one half frame at a time) permits morphometric analysis of dynamic alveolar function. The purpose of this report is to summarize our analyses of dynamic alveolar geometry during respiration and to relate these analyses to compliance hysteresis at the alveolar level. A preamble to this undertaking was a histologic correlation between what was observed in histologic section and what was observed in in Go microscopy to clarify the terms alveolus and alveolar sac as observed by the latter technique. Methods A.
LIGHT MICROSCOPY
The left pulmonary lobes of Sprague-Dawley rats weighing 250 g were inflated to a pressure of 20 cm HZ0 and immediately frozen with liquid nitrogen at - 196 “C. The lobes were then excised intact and fixed by freeze substitution (Staub and Storey, 1969). Sections from the anterior-inferior margin of these specimens, corresponding to the area of lung observed by in uiuo microscopy, were obtained by serial sectioning. Each section was 4 ~1thick. Maximum orthogonal alveolar diameters in two planes were determined for 31 alveoli and the mean alveolar diameter calculated. B.
IN WV0 MICROSCOPY TECHNIQUE
The anterior-inferior margin of the left lobe of 5 Sprague-Dawley rats was prepared for viewing with a videomicroscopy system by techniques described in previous reports (Daly and Norman? 1974; Daly et al., 1974). Briefly, ventilation via a tracheostomy was provided by a Harvard rodent respirator with a tidal volume of 4-6 ml at respiratory rate of 50 corresponding to the respiratory rate of the animals at rest. The airway pressure was continuously monitored at the outer end of the tracheostomy tube. For these studies, we utilized alveoli ventilated with positive pressure respiration both with an without 10 cm HZ0 positive end-expiratory pressure (PEEP). In a previous report PEEP was shown to increase both the total alveolar capacity or alveolar volume at peak inspiration, and alveolar functional residual capacity or alveolar volume at end expiration (Daly et al., 1974). In addition, PEEP was shown to increase the peak inspiratory pressure. Thus the effects of increased pressure and volume at total lung capacity and functional residual capacity on alveolar mechanics could be analyzed. A positive end-expiratory pressure
220
B. D.
T. DALY et al.
of 10 cm Hz0 was selected because it inflates the rat lung to nearly the top of its maximum pressure-volume (P-V) curve where atelectasis or recruitment and derecruitment should be eliminated. The videomicroscopy system consisted of a Wild-Heerbrug microscope with inverted epiobjective lenses, a Xenon arc light source, and a side-arm telescope connected in series with a television camera, image enhancer, time lapse video tape recorder (with stepped slow motion, stop action, and manual advance) and a video monitor. The inferior margin of the left lobe, stabilized by affixing pipe cleaners (approximately 0.5 cm apart) to its margins by medical adhesive, was approximated to a cover slip above the microscope objective. The traction and frictional resistance provided by the pipe cleaners prevented the left lobe from moving across the coverslip but did not inhibit 3-dimensional expansion and contraction of the lung enclosed within its margins. The area of lung studied was gently supported so that compression at the lung surface was minimized. Alveoli and alveolar sacs 1 to 1.5 mm below the pleural surface were visualized and their dynamic morphology recorded. c. ANALYSIS OF DYNAMIC
WALL
MOTION
For the investigation of the dynamics of alveolar wall motion, we applied the analysis of Hills (1971) for whole lung to individual alveoli. This analysis treats irreversibility or hysteresis as a function of surface energy, &. If the surface area of an alveolus is A, Es= yA, where y is the surface tension. Any loop in the pressurevolume curve could arise from an irreversibility in area apart from any irreversible dependence of surface tension on area, as assumed in attributing compliance hysteresis to surfactant alone. If geometric irreversibility can be demonstrated in an alveolus, then an irreversible relationship must exist between area and volume. It is possible the characteristics of geometric hysteresis curves could implicate the length-tension relationships of the alveolar wall and the stresses at alveolar interfaces as important determinants of hysteresis. The video recordings were replayed in slow motion and dynamic wall motion studied. Photographs of several alveoli and alveolar sacs were made from the video monitor at 0.033-see intervals to permit simultaneous comparisons between alveoli during different portions of the respiratory cycle. The alveolus from each rat which remained in the best focus throughout the respiratory cycle both with and without PEEP was selected for morphometric analysis. The technique utilized to stabilize the lung did not completely eliminate regional lung movement; it was, therefore, difficult to find more than a single alveolus in one field which was suitable for sequential morphometric analysis within a single breath. All alveoli, however, could be visually studied throughout a respiratory cycle by varying the focus. Two sets of paired points on the internal surfaces of the selected alveoli which could be followed accurately during respiration were identified. The distance or length (L) between these paired points (L, and L2) was measured directly from the video monitor. The videotapes were then advanced one frame at time throughout three breaths and
Fig. 1. A photomicrograph from a histologic section of rat lung frozen in uiuo and fixed by freeze substitution. The arrows point to folds in alveolar wails. x 480. Fig. 2. Each of these photographs was taken from images displayed on the television monitor, x 580. Schematic drawings of these images demonstrate the anatomic features of the alveoli. The solid areas in these schematic drawings represent alveolar walls; the stippled areas represent folds within the alveoli; and the clear areas depressions. (a) The arrow points to the wall of an alveolus. Its internal surface is comprised of many folds and depressions. These folds correspond to folds seen in histologic sections (fig. 1). The solid lines in the schematic drawing of this tigure connect paired points on the internal surface of the alveolus and represent typical L, and L, measurements.
(b) The same alveolus as in (a) 0.033 seconds later during uninterrupted respiration. Changes in the form and structure of the folds are evident. The solid lines in the schematic drawing demonstrate typical L 1 and L 2 measurements. The points of measurement correspond to identical points in the previous figure (2a).
(c) The portion of the mouth of an alveolus is seen directly below the O.
(d) The same alveolus as in (c) 0.033 seconds later during uninterrupted respiration. Changes in the form and structure of the folds are evident. The walls at the mouth of the alveolus have separated proportionately more than the remaining ·portions of the alveolar wall.
DYNAMIC
(e) Portions
of two alveolar
ALVEOLAR
MECHANICS
sacs are shown. The one on the left is comprised
3) and the one on the right of two alveoli (4 and 5).
223
of three alveoli (1, 2, and
224
B. D. T. DALY et ai.
the measurements repeated. Each measurement was repeated at 0.017~second intervals and an average of 65 measurements was made for each breath. These data were obtained from different alveoli by three different observers to eliminate possible bias in data acquisition. All measurements were entered on an IBM 35&155 computer via a time share terminal. L, and LZ measurements for each alveolusand each breath were then plotted separately by the computer as a function of time. A smooth curve was obtained by using a standard 5-point central difference smoothing technique. The smooth curves were then subtracted from the measured curves and the mean and standard deviation for the differences between all points on the measured and smooth curves were obtained for each breath. Tables were constructed by the computer for simultaneously obtained L, and L2 measurements for each breath utilizing every third point on the smooth curve. These time-paired L, and L2 points were then plotted. In order to identify artifacts introduced by the respirator, a rubber balloon was connected to the respirator, partially inflated and then further inflated and deflated by the respirator. Balloon wall motion was recorded on videotape. The changing dimensions of the balloon wall in a single plane were measured every 0.017 seconds and plotted as a function of time. Results
By light microscopy, the mean alveolar diameter from the exposed free edge of the left lobes of 250-g Sprague-Dawley rats inflated to 20 cm H,O pressure was 70.7 + 23.4 p (fig. 1). This measurement represents the average mean diameter since two orthogonal diameters from each of 31 alveoli were utilized in the computation. It is clear from these results that the structures illustrated in fig. 2ad represent alveoli and not alveolar sacs as we previously reported (Moreci and Norman, 1972, 1973; McNary et al., 1973; Daly et al., 1973; Daly er al., 1974). Mean alveolar diameter determined by video-microscopy techniques was 59.1+ 17.3 p. Alveolar sacs are illustrated in fig. 2e. These photographs were made directly from the television monitor and demonstrate not only the resolution obtained by these techniques but more importantly the variable internal structure of alveoli. Their internal surface is not smooth but rather punctuated by numerous folds and depressions which variably deform during respiration. The folds maintain their structural integrity from one breath to another. Figures 2a-b and 2cd show the changes in the internal structure of two alveoli which occurred 0.033 seconds apart. Figure 3 demonstrates the time changes in dimensions between one set of paired points measured across the internal surface of an alveolus during one breath. The smooth curve obtained by a central 5-point smoothing technique is also illustrated. Figure 4 shows similar curves obtained from a representative alveolus ventilated with 10 cm Hz0 PEEP. Several of the irregularities in these curves are due to respirator artifact. Figure 5 shows a time-dimensional curve obtained from a thin rubber balloon. The irregularities in this curve correspond to irregularities in the
225
Fig. 3. A time-dimensional curve showing the changes in the distances between one set of paired points across the internal surface of a normally ventilated alveolus throughout one breath. The points on the smooth curve selected for plotting hysteresis are shown as open circles and are connected by solid lines.
i TIME
110
&WW
Fig. 4. A timedimensional curve showing the changes in the distance between one set of paired points across the internal surface of an alveolus ventilated with positive end-expiratory pressure throughout one breath. The points on the smooth curve selected for plotting hysteresis are shown as open circles and are connected by solid lines.
TIME
&OMOI,
i
Fig. 5. A time-dimensional curve showing the changes in the distances across a rubber balloon in one plane as it is inflated and deflated by the respirator. The irregularities in the curve corresponded to
226
B. D. T. DALY
et ai.
above curves in time. While the changes in contour and irregularities of the curves from each set of paired points for each breath were similar, their relative contours changed from breath to breath. However, the changes were similar in each of the paired curves. L, and L, measurements for each breath were plotted parametrically. Frequently, the upslope and downslope of the resultant geometric hysteresis curves crossed each other at one or more points. In many instances, the reverses in these curves were abrupt. Although the curves for each breath in the same alveolus were different, the average slope of these curves was similar. Figure 6 illustrates a representative curve from a normally ventilated alveolus and fig. 7, one ventilated with PEEP. Several of these curves had multiple points of crossing at their base which represented points in time at the onset and termination of a breath. The upslope and downslope of the remaining portions of the curves had varying degrees of separation. This undoubtedly was affected by the actual location of the paired points IO-
sE 0
1 8b E
-I I-
& 1
6
6’ Fig. 6. A geometric rapid
reverses
hysteresis
curve for a normally
in this curve which are logically Maximum
7 LENCTN
point separation
Fig. 7. A geometric
hysteresis
b
ventilated
attributed
LEN&
curve for an alveolus
alveolus.
0.85 cm.
Note the sudden forces within
changes
the alveolar
and wall.
on the x axis and is I. I5 cm.
I- 2
ventilated
than that seen in fig. 6, and maximum
9
to mechanical
on this curve occurs
ecurve is smaller
I -cm
with positive
point
separation
end expiratory occurs
pressure.
The
on the y axis and
is
DYNAMIC
ALVEOLAR
227
MECHANICS
relative to the alveolar wall. The loops ofnormally ventilated alveoli were significantly longer than those for alveoli ventilated with PEEP with a single exception in the PEEP group. On reviewing these tapes, we discovered that one point from each set of paired points measured on the internal surface of this alveolar wall was adjacent to the mouth of an alveolus where it opened into an alveolar sac. Relatively greater wall motion was observed but not quantitated at similar points in other alveoli. Table 1 shows the maximum distance separating points on either the horizontal or vertical axis for each hysteresis curve in both the normally ventilated alveoli and those ventilated with positive end-expiratory pressure as well as the mean and standard deviation of the distance separating all points between the measured and smooth curves. True separation of any two points on these hysteresis curves demonstrated geometric irreversibility within the alveolar wall. Such separation was present for every curve in both groups. However, the separation was TABLE.1 Maximum
Alveoli ventilated
without
Alveolus-
Distance
breath
(cm**)
hysteresis point dikrential Alveoli ventilated
PEEP
Error S -.--
I-1
with PEEP
Alveolus-
Distance
breath
(cm) --.-
.--
PI
0.85
0.084 + 0.075
--1
Error
1.50
0.120*0.121
2
2.80
0.173kO.173
2
I.25
0.087 f 0.072
3
1.20
0.147f0.139
3
2.14
0.098 & 0.092 0.100+0.148
II -
I
0.90
0.148+0.113
- I
I .25
2
1.75
0.153kO.129
2
I.75
0.078 f 0.072
2
1.15
0.148+0.118
3
I.20
0.07 I + 0.060
PII’
111 - I
2.00
0.099 f 0.105
PI11 - 1
0.85
0.069 f 0.062
2
1.20
0.078 k 0.089
2
0.45
0.07 I f 0.082
3
2.00
0.077 k 0.084
3
0.70
0.067 f 0.065
IV - I
1.15
0.090 + 0.084
PIV - I
0.55
0.084 f 0.08 I
2
I.60
0.102 +_0.079
2
0.40
0.079 f 0.088
3
1.45
0.087 k 0.066
3
0.45
0.077 f 0.059
I
0.90
0.107+0.115
2
1.40
0.104kO.123
3
0.90
0.088 + 0.103
i
1.46
P < 0.05
SD
0.52
V’ -
1.01 0.60
* In rat V we were unable to follow points on the internal surfaces of an alveolus sequentially out repiration l
* All points were measured off the TV monitor 8 k+S.D.
for distance separating
in cm and plotted with the same units.
points on the smooth and measured curves for L, and L, of each
breath.
’
through-
with PEEP.
One point of each set of paired points of PI1 was at the mouth of an alveolus.
228
B. D. -I-. DALY et id.
significantly less (PC 0.05) in the PEEP group. Discussion In oiuo microscopy of the lung is not a new technique. Olkin and Joannides (1930), using a 6-volt light source and a microscope with 60 x magnification accurately described the living anatomy of alveoli. Since that time, other reports of in uiuo studies have appeared but these have mostly been devoted to the pulmonary microcirculation or to refinements in technique. DeAlva and Rainer (1963), Wagner and Filley ( 1965) and Sherman et al. (197 1) showed the in uiuo appearance of pulmonary alveoli but did not describe their dynamic morphology. The first in uiuo morphometric analysis of alveoli was performed by Wagner ( 1969). Although he reported quantitative measurements on alveolar diameters, he did not relate these to respiration. Moreci and Norman (1972, 1973) described the effects of positive pressure respiration on alvelar diameters of rat lung in inspiration and expiration and detailed their morphology. However, the terminal respiratory units visualized by incident light dark-field illumination were thought to represent alveolar sacs because of the many complete subdivisions observed within these terminal structures. In subsequent reports, the term “alveolar sac” was used in morphometric analyses of these structures (McNary et al., 1973; Daly et al., 1973; Daly et al., 1974). The measurements for “alveolar sacs”, however, correlate with measurements of rat alveoli obtained by others on fixed tissue (Tenney and Remmers, 1963; Macklin and Hartroft, 1943; Burri et al., 1974). The present investigation compares for the first time what is observed by histologic section and in uiuo microscopy. A rapid freezing technique using liquid nitrogen and freeze substitution was utilized to minimize tissue distortion in the histologic preparations. The animals used for these studies were prepared in the same manner as those utilized for in uiuo microscopy and comparable areas of lung were selected for study. Since the exposed lung was frozen and only the superficial alveoli were examined, liquid nitrogen was utilized in preference to liquid propane because of the hazards involved in the latter technique (Staub and Storey, 1969). In the histologic sections, alveolar diameters measured 70.7 k23.4 ,U and by in uiuo microscopy, 51.1 f. 17.3 p. These values are comparable. The lower figure obtained by in uiuo microscopy probably resulted from a lower mean inflation pressure. The lungs utilized for histologic study were inflated to 20 cm of H,O whereas the peak inflation pressure utilized in the in uiuo studies varied from 15 to 20 cm Hz0 (Daly et al., 1974). It is clear from these studies that the terminal respiratory units visualized in fig. 2acl represent alveoli and those in fig. 2e represent alveolar sacs. Our time lapse studies of alveolar wall motion have demonstrated the dynamic changes occurring within the alveolar wall with respiration. The folds observed in the walls of alveoli in histologic preparations would appear from our studies to represent the counterparts of the folds observed in uiuo. If this is true, these folds are not haphazard artifacts of tissue preparation but have both form and structure. The observation that wall motion at the mouths of alveoli is greater than wall
DYNAMIC
ALVEOLAR
MECHANICS
229
motion in other parts of alveoli indirectly supports the work of Macklin (1950) who expressed the view that the main increase in volume during inflation occurs in alveolar ducts. While we were unable to focus on alveolar ducts per se, we did observe relatively greater wall motion at the mouths of alveoli where two or three appeared together to form an alveolar sac. It is clear from our analyses that geometric irreversibility is one characteristic of alveolar wall motion. The sharp reverses and rapid changes in several of the observed geometric hysteresis curves are logically attributed,’ to mechanical wall properties and suggest that elastic forces within the alveolar wall or interface forces from adjacent alveoli are important in terms of local wall changes, Such forces were demonstrated in a mechanical lung model (Mead et al., 1970). Another aspect of dynamic irreversibility is the nonrepetitive wall movement observed in the timedimensional curves from breath to breath. Any error in the methods introduced by smoothing the curves is accounted for in table 1. The frequency with which the points for each curve were obtained and the acquisition of the points by three different observers eliminates any error introduced by observer bias. Although it is possible that the original points selected for measurement could have shifted within the alveolar wall as it thickened or stretched with respiration or changed its plane of focus slightly, these changes should have occurred equally for both dimensions measured. The time-dimensional changes recorded for a rubber balloon accounts for any respirator artifact. The report by Gil and Weibel (1972) supports these conclusions. Figure 11 in this report demonstrates irreversibility in a three-dimensional plot of alveolar surface area uerms both pressure and volume. Although Es= ?A, the gas in the alveolus contributes - PdV (inflation energy) to the total energy within the alveolar wall (P is the inflation pressure and V is the alveolar volume). The instantaneous wall energy within the alveolus can be expressed as dE = YdA- PdV. In a previous report (Daly et al., 1974) ventilation with a PEEP of 10 cm Hz0 was shown to inflate the rat lung to nearly the top of its P-V curves. Since A is proportional to volume to the 2/3 power, one could expect E, to decrease as V increased. This is in fact what was observed. At a PEEP. of 10 cm H20, geometric hysteresis was still observed but was less (PC 0.05) than that observed in the alveoli ventilated without PEEP. Although one aspect of irreversibility is recruitment and derecruitment, at this level of PEEP, alveolar recruitment and derecruitment should be minimally present if not absent. These studies demonstrate the dynamic nature of alveolar wall motion and the irreversibility of alveolar expansion and contraction. Thig can be explained by an irreversibility in alveolar area apart from any irreversible dependence on surface tension as assumed in attributing compliance hysteresis to surfactant alone. Recruitment and derecruitment of alveoli cannot completely explain this phenomenon. The characteristics of our alveolar geometric hysteresis curves both with and without PEEP suggest that the length-tension properties of elastic tissues, their distribution within the alveolar wall, and the interface forces from adjacent alveoli are important determinants of local alveolar wall motion and pulmonary hysteresis.
230
B. D. T. DALY et d.
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Clements, J. A., E. S. Brown and R. P. Johnson (1958). Pulmonary surface tension and the mucus lining of the lungs: Some theoretical considerations. J. Appl. Physiol. 12: 262-268. Daly, B. D. T.. C. H. Edmonds and J. C. Norman (1973). In uioo alveolar morphometrics with positive end expiratory pressure. Surg. Forum XXIV: 217-219. Daly, B. D. T. and J. C. Norman (1974). Alveolar morphometrics 3: Preliminary results with in uiuo videomicroscopy. Chest 65: 67-68. Daly, B. D. T., D. A. Hughes and J. C. Norman (1974). Alveolar morphometrics: Effects of positive end expiratory pressure. Surgery 76: 624629. DeAlva, W. E. and W. G. Rainer (1963). A method of high speed in uiuo pulmonary microcinematography under physiologic conditions. Angiology 14: 160-164. Gil, J. and E. R. Weibe.1 (1969/‘1970). Improvements in demonstration of lining layer of lung alveoli by electron microscopy. Respir. Physiol. 8: 13-36. Gil, J. (1971). Ultrastructure of lung fixed under physiologically defined conditions. Arch. Inc. Med. 127: 896902. Gil, J. and E. R. Weibel(l972). Morphological study of pressure-volume hysteresis in rat lungs tixed by vascular perfusion. Respir. Phvsiol. IS: 190-213. Hills_ B. A. (1971). Geometric irreversibility and compliance hysteresis in the lung. Respir. Physiol. 13: SOdI. Macklin, C. C. and W. S. Hartroft (1943). Extramural Report to the Canadian Subcommittee on Physiological Aspects of Chemical Warfare. C.P. 35. Macklin, C. C. (1950). Alveoli of mammalian lung. Anatomical study with clinical correlation. Proc. Inst. Med. Chic. 18: 78-95. McNary. W. F., H. S. Berman, B. Daly and J. C. Norman (1973). The use of videotape to record and review data obtained by incident light dark-field microscopy of the living lung. Bib!. Anat. II : 4449. Mead, J., J. L. Whittenberger and E. P. Radford (1957). Surface tension as a factor in pulmonary volume-pressure hysteresis. J. Appl. Physiol. 10: 191-196. Mead, J., T. Takishima and D. Leith (1970). Stress distribution in lungs: A model of pulmonary elasticity. J. Appl. Physiol. 28: 596-607.
Moreci, A. P. and J. C. Norman (1972). Q uantitative and qualitative changes in morphology of alveolar sacs with positive pressure respiration. Proc. Sot. Expfl. Biol. Med. 141: 318-321. Moreci. A. P. and J. C. Norman (1973). Measurements of alveolar sac diameter by incident-light photomicrography. Ann. Thorac. Surg. 15: 179-186. Olkin, D. M. and M. Joannides (1930). Capillaroscopic appearance of the pulmonary alveoli in the living dog. Anat. Rec. 45: 121-127. Pattle, R. E. (1955). Properties, function and origin of the alveolar lining layer. Nature (London) 175: 1125. Pierce, J. A., J. B. Hocott and W. F. Helley (1961). Elastic properties and the geometry of the lungs. J. C/in. Inuesr. 40: 1515-1524. Radford, E. P. (1957). Recent studies of mechanical properties of mammalian lungs. In: Tissue Elasticity, edited by J. W. Remington. Washington, American Physiology Society, pp. 177-190. Radford, E. P. (1964). Static mechanical properties of mammalian lungs. In: XXII Intern. Congr. Sci., Vol. I, pp. 275-280. Sherman, H.. S. Klausner and W. A. Cook (1971). Incident dark-field illumination: A new method for microcirculatory study. Angialogy 22: 295-303.
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MECHANICS
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