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Mechanical forces contribute to neonatal lung growth: The influence of altered diaphragm function in piglets
Mechanical Forces Contribute to Neonatal Lung Growth: The Influence of Altered Diaphragm Function in Piglets ByMitchell
R. Price, Mark E. Galantowicz,
and Charles J.H. Stolar
New York, New York 0 Neonatal lung growth is controlled in part by mechanical forces. Altered mechanical forces precipitated by phrenectomy or prosthetic replacement of the diaphragm result in altered thoracic volume relationships, which, in turn, change lung distending pressures and or thoracic volume. These effects might contribute to regional lung growth. We postulated a relationship between altered thoracic mechanical forces and changes in lung growth and asked if altered diaphragm function influenced regional lung growth. Piglets (28d. 7-8kg). were assigned to left transthoracic phrenectomy (P), prosthetic diaphragm replacement (PDR), or sham (S), (n = 8). After a mean 10 days, piglets were studied with tracheostomy and regional pleural pressure transducers. Integrated lung volumes (LV) were recorded with intrapleural pressure (Pip). Dynamic compliance (Cdyn) was calculated (dV/dP). After sacrifice continuous pressure volume (P/V) CUNBS were generated. Lungs were then cut into 4 quadrants based on relationship to R/L bronchus and processed for DNA content and total protein indexes. Analysis of data were made within and between groups. Body weight and gain were similar in all. LV, Pip, Cdyn, and P/V were not significantly different in PDR and P compared with S. Pip differences between thoracic regions within each group were significant for PDR and showed LU c RU, LL < RL (P c .05). RU and RL Pip in the PDR group were the same as S. Pip in the P group were decreased in the RU, LU, and LL but only the LL approached significance. Whole lung wet weights were decreased (P c .05) in P compared to PDR and S. P showed significant reduction (P < .05) in total quadrant DNA and protein, and wet weights. (P < .05) in RLQ and LLQ compared with S and PDR. P showed reduction in dry weight of LLQ compared with PDR or S (P < .05). Total protein/DNA ratio was similar in all groups. (1) Impaired diaphragm function alters thoracic volume relationships and decreases regional pleural pressures. (2) Phrenectomy decreases lung cell size (protein) and number (DNA) in lung quadrants closest to the impaired diaphragm compared to prosthetic and sham. (3) Prosthetic diaphragm minimizes DNA/protein compromise despite decreased regional pleural pressures. (4) Restored thoracic volume contributes to neonatal lung growth. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Postnatal lung growth.
From the Department of Pediatric Suqety, Babies Hospital, New York, NY Sponsored in part by The Charles Edison Fund, Orange, NJ, and The Anya Fund, Armor& NY Presented at the 22nd Annual Meeting of the American Pediatric SurgicalAssociation, Lake Buena Vi&a, Florida, May 15-18, 1991. Address reprint requests to Charles J.H. Stolar, MD, Babies Hospital, Room 203N, 3959 Broadway, New York, NY10032. Copyright o I992 by W. B. Saunders Company 0022-3468/92/2703-OO19$03.OOlO
376
L
UNG GROWTH is controlled at least in part by mechanical forces. Changes in thoracic vo1ume1-4 and unilateral diaphragm paralysis’-’ are reported to have a significant effect on respiratory mechanics and compensatory lung growth. In fetal models, phrenic nerve section results in a reduction in both lung growth and alveolar maturation.‘,’ These studies suggest that lung distending pressure (increasing thoracic volume relative to maximum lung volume) and thoracic volume act as regulators of normal postnatal lung growth. Consequently, we speculated that altered mechanical forces produced by phrenectomy or prosthetic replacement of the diaphragm might result in altered thoracic volume relationships, which would in turn, change lung distending pressures and or thoracic volume, resulting in altered regional lung growth. We postulated that there is a relationship between altered mechanical forces and specific changes in regional lung growth, and hypothesized that if this is indeed true, then altering the function of a major muscle of respiration, the diaphragm, should influence regional lung growth. MATERIALS
AND METHODS
Eighteen neonatal piglets (aged 28 days, weighing 7 to 8 kg) were assigned equally to one of three groups: left transthoracic phrenectomy, phrenectomy and diaphragm replacement with a noncompliant silastic patch, or sham thoracotomy. The animals had fluoroscopy before experimentation to document the position of the mediastinum and to note the respiratory excursion of the diaphragm on the involved side. After a mean of 10 days recovery, anesthetized spontaneously breathing piglets were studied supine after preparation with occlusive tracheostomy, placement of esophageal balloon (0.1 mL air inflation) in the midthoracic region, and insertion of specifically designed regional intrapleural pressure monitors (2nd and 8th ICS/MAL) coupled to Validyne pressure transducers (Validyne Engineering Corp, Northridge, CA). Animals were sedated at intervals (every 20 to 25 minutes) with sodium pentobarbital (5 mg/kg) and ketamine (5 to 10 mgikg) sufficient for sedation while enabling spontaneous respirations. Real-time integrated lung volumes were measured by a Hans Rudolf pneumotachograph (Hans Rudolph, Inc, Kansas City, MO) and recorded simultaneously with both esophageal pressure and regional intrapleural pressure on an analog data physiograph. Dynamic compliance (dV/dP) was also calculated from these data. Physiological data were analyzed by an in-line data software acquisition program. The animals were then killed and pressure/volume hysteresis curves were generated by inflating the lungs to a pressure of 30 cm H,O and then extracting sequential 5mL increments of air while recording the pressure at each point.
JournalofPediatric
Surgery, Vol27, No 3 (March), 1992: pp 376-381
377
MECHANICAL FORCES AND NEONATAL LUNG GROWTH
The piglet’s sternum was then opened and the lungs were removed en bloc. They were then divided into four reproducible, anatomic quadrants based on their relationship to the right/left mainstem bronchus (right upper quadrant [RUQ], right lower quadrant [RLQ], left upper quadrant [LUQ], and left lower quadrant [LUQ]. Pulmonary tissue in each quadrant was then assayed for total DNA content (Fleck and Monro technique”‘) and for total protein content (Lowry et al technique”). The results were then averaged for each particular group by quadrant and comparisons were made between the three groups based on these data points. Statistical analysis was carried out via analysis of variance and individual means were tested using Duncan Multiple Range Test.
251
\
Phrenectomy _______Sham ----Patch
RESULTS
Original body weight and weight gain were similar between the three groups. Fluoroscopy showed: (1) sham group: normal diaphragmatic excursion and fixed mediastinum; (2) phrenectomy group: paradoxical motion of an eventrated diaphragm, minimal movement of the mediastinum, and visual loss of thoracic volume on the involved side; and (3) patch group: tethered grossly noncompliant patch with slight paradoxical motion, a fixed mediastinum, and restoration of thoracic volume on the involved side. Pulmona y Function Data Global lung volumes, dynamic compliance (Cdyn) and pressure-volume (P/V) curves as well as regional pleural pressures were reduced in both the phrenectomy and the patch groups compared with the sham controls (Table 1, Fig l), but variations noted among the three groups were not statistically significant. Pleural pressure data (Fig 2) comparing regional differences within the thoracic cavity in each of the study groups showed the following: (1) the sham group exhibited equivalent pleural pressure readings in all four thoracic regions-RUQ(13.107 k 2.95 cm H20), RLQ(13.682 + 3.06 cm HzO), LUQ(13.125 -+ 3.01 cm H,O), LLQ(13.273 k 1.98 cm H,O) (Fig 2); and (2) the phrenectomy group had decreased pleural pressures in the RUQ(11.652 ~fr. 4.11 cm H,O), LUQ(10.460 k 2.87 cm H,O), and LLQ(11.171 -+ 5.61 cm HzO) (Fig 2). The differences among the four regions within this group were not statistically significant. The pleural pressures in the LLQ appeared to be decreased compared with the
0
100
400
200 300 Volume (cc)
Fig 1. Closed chest P/V hysteresis curves generated for the three experimental groups (P = NS).
RLQ but because of the large variability within this group this difference only approached significance. The patch group also exhibited decreased pleural pressures but were limited to the LUQ(ll .OOO+ 4.33 cm HzO) and LLQ(10.344 + 4.61 cm HzO) (Fig 2). The decreases in the LUQ and the LLQ compared with the RUQ(13.423 k 5.38 cm HzO) and RLQ(13.330 + 4.74 cm HzO) were significant (P < .05). The right-sided pressures in the patch group were equivalent to the right-sided pressures found in the sham control. WetlDy Weight, DNAlProtein indexes Total lung wet weights were decreased (P < .05) in the phrenectomy group (74.81 5 7 g) compared with both the sham group (104.23 5 17 g) and the patch group (106.67 + 22 g). Only the phrenectomy group
\_,
---\-. SHAM
PHRENECTOMY
‘A-
PATCH
Table 1. Pulmonary Function Tests
Lung volume
Sham
Phrenectomy
Patch
60.66 f 22
50.06 + 18
42.50 ? 6
Dynamic compliance Right
2.647
3.074
1.949
Left
2.028
2.098
1.919
NOTE. Data given as mean t- SD.
Fig 2. Regional pleural pressures schematically depicted for each experimental group. There was no significant difference in pleural pressure among the sham, phrenectomy, or patch groups. Differences between thoracic regions within each experimental group was significant (“P < .05) for patch LUD < RUQ and LLQ -z RLQ. The patch restored thoracic volume relationships (defect isolated to hemkhorax) compared with the phrenectomy group (three-quadrant effect). Values are expressed in cm H,O.
378
PRICE, GALANTOWICZ,
showed significant reductions (P < .OS) in wet weight (Fig 3), dry weight (Fig 4), total DNA content (Fig 5), and total protein content (Fig 6). Neither the sham nor the patch groups showed significant reductions in the above indexes and, in fact, showed consistently similar results. The phrenectomy group showed a significant decrease (P < .05) in total quadrant DNA, total quadrant protein, and wet weights in both the RLQ and LLQ when compared with either sham or patch groups. The phrenectomy group also demonstrated significant reduction (P < .05) in the dry weight of the LLQ(3.43 & 0.94 g) compared with patch and sham groups. Dry weight/wet weight and total protein per gram dry weight and DNA per gram dry weight ratios were not found to be statistically different between the three groups. DISCUSSION
We postulated a relationship between altered mechanical forces and specific changes in regional lung growth hypothesizing that altered function of a major muscle of respiration, the diaphragm, could influence regional lung growth. A previous study of unilateral phrenectomy in cats and piglets” showed unchanged total growth of both lungs combined with an inability to differentiate between decreased growth on the ipsilateral side, increased growth on the contralateral side, or a combination of both. This experiment underscores the importance of evaluating interregional differences in an attempt to isolate specific changes in regional lung growth. By monitoring different regions of the thoracic cavity (regional pleural monitors), we tried to match a specific change in pleural pressure (phrenectomy or nonmobile diaphragm) with a concurrent change in regional lung growth. Phrenectomy causes changes in thoracic configuration, vo1ume,13 and regional intrathoracic pleural pressure.14 The cranial/dorsal pleural pressures increase while the caudalkentral pleural pressures
SHAM
PHRENECTOMY
Y%ir
Fig 3. Schematic of quadrant wet weights (g) in the three experimental groups. Phrenectomy showed signincant decrease (*P c .Ol) in quadrant wet weights in the RLQ and LLQ compared wlth sham and patch groups.
SHAM
PHRENECTOMY
AND STOLAR
7tkii-
Fig 4. Schematic of quadrant dry weights (g) in the three experimental groups. The phrenectomy group showed significant decrease (V c .05) in quadrant dry weights in the LLD compared with the sham and patch groups.
decrease as the scalenes, intercostal, and other accessory muscles of respiration replaced the diaphragm as muscles of respiration. If, as we postulated, lung distending pressure is a component of lung growth, then we would expect the greatest loss of growth to be noted in the area closest to the diaphragm. We would further anticipate diminished growth on the ipsilatera1 side secondary to a loss of thoracic volume. The patch animals with a loss of lung distending pressure (nonmuscular diaphragm), but restitution of thoracic volume should have growth between the two groups. The DNA and total protein data should parallel changes seen in the interregional pleural pressures. Pleural Pressure
These results confirmed differences in interregional transpulmonary pressures within both the patch and phrenectomy group. The phrenectomy group showed a three-quadrant effect with the RUQ joining the LUQ and LLQ in being affected by the left-sided phrenectomy. Also, the LUQ showed a reduction in pleural pressure and not an increase or stabilization as anticipated. The fact that the phrenectomy group did not show statistically significant differences in interregional pleural pressures may be attributed to this three-quadrant affect, although fluoroscopy showed only slight move-
SHAM
A
PHRENECTOMY
-
PATCH
Fig 5. Schematic of quadrant DNA (mg) in the three experimental groups. Phrenectomy showed significant decrease (*P c .05) in RLQ and LLQ DNA compared with the sham and patch groups.
379
MECHANICAL FORCES AND NEONATAL LUNG GROWTH
SHAM
A
PHRENECTOMY
-
PATCH
Fig 6. Schematic of quadrant protein content (mg) in the three experimental groups. The phrenectomy showed significant decrease (*P c ,051 in RLQ and LLQ protein compared with the sham and patch groups.
ment of the mediastinum in this surgical group. The LLQ pressures were prevented from reaching significance because of the large variability within this one particular group. The patch group also showed a reduction in LUQ and LLQ pleural pressures. This was expected, but of interest is the restoration of the RUQ pleural pressure mimicking that of the right side of the sham animal and isolating the defect to the left side only. The patch animal actually appeared to follow the rules of D’Angelo et all4 in that the cranial segments bilaterally recorded higher pleural pressures than the caudal segments. Biological Assays
The results of the biological assays in the phrenectomy group showed that the quadrants closest to the impaired diaphragm were affected the most and demonstrated a statistically significant reduction in growth compared with the sham animals. Both lower quadrants were affected despite the fact that the pleural pressures on the RLQ were not decreased significantly. It may be that the sensitivity of biological assays is greater than that of the intrapleural monitors. In addition, the relationship between the muscular action of the diaphragm (mechanical forces) and specific changes in regional lung growth, while still valid, raises the possibility that other factors and mediators may be at work. Although the diaphragm is divided by the mediastinum and is supplied by two distinct nerves, impairment of any one half may greatly effect the contralateral side in terms of lung
distending pressure, diaphragmatic excursion, and/or thoracic volume. Whereas these transpulmonary pleural pressures may appear to indicate normal functioning mechanical forces, a more sensitive test (biological assays) shows otherwise. The loss of cell number (DNA) without a change in mean cell size (total protein/DNA) demonstrates a loss of mitotic figures and is reminiscent of cellular hyperplasia and compensatory lung growth.‘” We speculate that forces invoked in compensatory lung growth may be involved in normal postnatal lung growth and that these forces were altered in our study. The patch group showed no reduction in interquadrant wet weight, dry weight, DNA, and total protein when compared with the sham group; however, the pleural pressures were significantly decreased in the affected ipsilateral hemithorax. The mechanics of the patch spared the loss of growth. Because empty thoracic space serves as a stimulus to lung growth (ie, compensatory hyperplasia postpneumonectomy),‘5 a loss of thoracic volume and paradoxical motion of the diaphragm could retard normal lung growth on the effected side. The mechanism for this could involve one or more mechanical forces such as lung distending pressure, thoracic volume, and/or normal diaphragmatic excursion. The presence of a minimally compliant patch in a closed hemithorax undergoing negative pressure during inspiration may generate some level of lung distending pressure. This, coupled with some restoration of thoracic volume and fixed diaphragmatic movement, may be enough to minimize a loss of growth. We speculate that postnatal lung growth is governed in part by parenchymal stretch caused by lung distending pressure, but that an even more important role may be played by either thoracic volume restoration and/or normal diaphragmatic excursion. This may help to explain the loss of growth in the lower quadrants in the phrenectomy group and the apparent normalization of growth in the patch animals despite the fact that lung distending pressure (transpulmonary pressure) was decreased in both. Further study is needed to clearly elucidate the role of these factors in neonatal lung growth.
REFERENCES 1. Dunnill M: Quantitative observations on the anatomy of chronic non-specific lung disease. Med Thorax 22:261-269, 1965 2. Davies G, Reid L: Effect of scoliosis on growth of alveoli and pulmonary arteries and on the right ventricle. Arch Dis Child 46:623-632,197l 3. Cowan MJ, Crystal RG: Lung growth after unilateral pneumonectomy: Quantitation of collagen synthesis and content. Am Rev Respir Dis 111:267-277.1975
4. Simnett JD: Stimulation of cell division following unilateral collapse of the lung. Anat Ret 180:681-686. 1974 5. Arborelius M, Lilja B, Senyk J: Regional and total lung function studies in patients with hemidiaphragmatic paralysis. Respir 321253-264, 1975 6. Ridyard JB, Stewart RM: Regional lung function in unilateral diaphragmatic paralysis. Thorax 31:438-442, 1976 7. Easton PA, Fleetham JA, De La Rocha A, et al: Respiratory
380
PRICE, GALANTOWICZ,
function after paralysis of the right hemidiaphragm. Am Rev Respir Dis 127:125-128,1983 8. Wigglesworth JS, Desai R: Effects on lung growth of cervical cord section in the rabbit fetus. Ear Hum Dev 3:51-651979 9. Nagai A, Thurlbeck WM, Jansen AH, et al: The effect of chronic biphrenectomy on lung growth and maturation in fetal lambs. Am Rev Respir Dis 137:167-172, 1988 10. Fleck A, Monro HN: The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim Biophys Acta 55:571-583, 1962 11. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275,195l
AND STOLAR
12. Mansell AL, Velasquez-Rojas J, Sillos M, et al: Diaphragmatic activity is a determinant of postnatal lung growth. J Appl Physiol61:1098-1103, 1986
13. De Troyer A, Kelly S: Chest wall mechanics in dogs with acute diaphragm paralysis. J Appl Physiol53:373-379,1982 14. D’Angelo E, Sant’Ambrogio G, Agostoni E: Effect of diaphragm activity or paralysis on distribution of pleural pressure. J Appl Physiol37:311-315, 1974 15. Cagle PT, Thurlbeck WM: Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 138:1314-1326,1988
Discussion J.A. Huller (Baltimore, MD): I think the juxtaposi-
tion of these two papers this morning has great pertinence to all of us who care for babies with diaphragmatic hernias. Because I suspect that because we are so pressed in the care of babies who are dying with diaphragmatic hernia and, therefore, carrying out so many clinical trials, we need to come back to this base-what’s going on in the chest. This paper is particularly important because it reminds us that we still have a lot to do in understanding the pathophysiology of the diaphragmatic hernia and understanding better the growth factors related to the lung. The authors have chosen a neonatal subject, the piglet, and I want to focus some of my comments and questions on whether they have yet transferred this to the fetal animal. Because this is where we really need to be looking at the factors associated with not only the development of the diaphragmatic hernia, which remains an enigma, but also which factors result in the abnormal development of the lungs associated with it. There is some good background information and you saw in the initial slide some reference to that neonatal work. But the rapidity of lung growth and the maturation of the pulmonary vascular bed clearly occurs long before there is a piglet, I don’t know what a fetal piglet is called other than that, and it seems to me that focus needs now to be addressed. For example, we know that if a balloon is put in the fetal chest, this will interfere with lung growth and development. Dr Michael Harrison and his associates carried out that study. We had carried out similar studies earlier. We also know from other work that if you ligate the fetal bronchus, the lung does not grow and yet the mechanisms for that remain unclear. Is obstructing drainage from the lung responsible for interfering the changes in the histology and if so how? We need to address the problem itself rather than looking at the clinical manifestations of it. We must continue our clinical trials. But the pessimistic results
from Boston Children’s simply underlines the importance of this paper, which is going back to the laboratory. I would like to ask just a couple of questions and ask the authors if they would project for us some of their thoughts based upon this neonatal model. Do you think it’s possible that the diaphragmatic defect occurs because the lung doesn’t develop? We don’t know the factors responsible for closure of the pleuroperitoneal canal but it may simply be a result of the fact that it isn’t a normal lung to begin with. We know that babies as well as fetal animals who do not have normal pulmonary movement, that is respiratory movement in utero, are often born with major pulmonary abnormalities and indeed other systems may be involved. What is the function of respiratory movement in the developing lung? And does it having something to do with the diaphragm? And, if so, is the development of the diaphragm itself secondary to abnormalities in pulmonary function? I know you have thought about this. Have you also carried out your studies yet in fetal animals? Can you share that with us yet? Or is that going to be on next year’s program? H. F&ton (Knoxville, TN): I’m sort of surprised where Alex focused because my simplistic mind saw this more as a study of eventration and I think what I see is an elegant study showing us why, perhaps, we have to plicate diaphragms. This issue has been debated in this forum in the past and I would just bring it out of the realm of the diaphragmatic hernia and say it looks to me like a very nice model for eventration and tells us a good bit about why sometimes you can’t get babies who have eventration of the diaphragm off ventilators. i&KPrice (response): Thank you Drs Haller and Filston for your comments. Our work involves postnatal rather than fetal lung growth; specifically, growth as it relates to the actions of the respiratory muscles. Fetal and postnatal lung growth are two distinct
MECHANICAL
FORCES AND NEONATAL
LUNG GROWTH
entities and attempts at comparing the two are problematic. We can only speculate on the mediators of fetal lung growth and agree with you that the mechanisms as they pertain to congenital diaphragmatic hernia or any other in utero space occupying thoracic lesion are unclear at this juncture. Certainly, thoracic volume, respiratory movement, and humoral factors all appear to play a substantial role. What we can comment on is that our work suggests that postnatally, particularly in congenital diaphragmatic hernia patients, placement of a noncompliant patch or other attempts to restore thoracic volume may be
381
beneficial as an impetus toward improved lung growth on the involved side. Our only reservation about this last statement is that this study was performed on a healthy piglet model and we must be careful when trying to extrapolate animal findings to diseased human lungs. The concept of thoracic volume as a principal mediator of postnatal lung growth is an exciting one, but much work needs to be done in the areas of lung architecture, humoral mediators, and the role of respiratory excursion before this theory can be put into clinical practice.
R. Price, Mark E. Galantowicz,
and Charles J.H. Stolar
New York, New York 0 Neonatal lung growth is controlled in part by mechanical forces. Altered mechanical forces precipitated by phrenectomy or prosthetic replacement of the diaphragm result in altered thoracic volume relationships, which, in turn, change lung distending pressures and or thoracic volume. These effects might contribute to regional lung growth. We postulated a relationship between altered thoracic mechanical forces and changes in lung growth and asked if altered diaphragm function influenced regional lung growth. Piglets (28d. 7-8kg). were assigned to left transthoracic phrenectomy (P), prosthetic diaphragm replacement (PDR), or sham (S), (n = 8). After a mean 10 days, piglets were studied with tracheostomy and regional pleural pressure transducers. Integrated lung volumes (LV) were recorded with intrapleural pressure (Pip). Dynamic compliance (Cdyn) was calculated (dV/dP). After sacrifice continuous pressure volume (P/V) CUNBS were generated. Lungs were then cut into 4 quadrants based on relationship to R/L bronchus and processed for DNA content and total protein indexes. Analysis of data were made within and between groups. Body weight and gain were similar in all. LV, Pip, Cdyn, and P/V were not significantly different in PDR and P compared with S. Pip differences between thoracic regions within each group were significant for PDR and showed LU c RU, LL < RL (P c .05). RU and RL Pip in the PDR group were the same as S. Pip in the P group were decreased in the RU, LU, and LL but only the LL approached significance. Whole lung wet weights were decreased (P c .05) in P compared to PDR and S. P showed significant reduction (P < .05) in total quadrant DNA and protein, and wet weights. (P < .05) in RLQ and LLQ compared with S and PDR. P showed reduction in dry weight of LLQ compared with PDR or S (P < .05). Total protein/DNA ratio was similar in all groups. (1) Impaired diaphragm function alters thoracic volume relationships and decreases regional pleural pressures. (2) Phrenectomy decreases lung cell size (protein) and number (DNA) in lung quadrants closest to the impaired diaphragm compared to prosthetic and sham. (3) Prosthetic diaphragm minimizes DNA/protein compromise despite decreased regional pleural pressures. (4) Restored thoracic volume contributes to neonatal lung growth. Copyright o 1992 by W.B. Saunders Company INDEX WORDS:
Postnatal lung growth.
From the Department of Pediatric Suqety, Babies Hospital, New York, NY Sponsored in part by The Charles Edison Fund, Orange, NJ, and The Anya Fund, Armor& NY Presented at the 22nd Annual Meeting of the American Pediatric SurgicalAssociation, Lake Buena Vi&a, Florida, May 15-18, 1991. Address reprint requests to Charles J.H. Stolar, MD, Babies Hospital, Room 203N, 3959 Broadway, New York, NY10032. Copyright o I992 by W. B. Saunders Company 0022-3468/92/2703-OO19$03.OOlO
376
L
UNG GROWTH is controlled at least in part by mechanical forces. Changes in thoracic vo1ume1-4 and unilateral diaphragm paralysis’-’ are reported to have a significant effect on respiratory mechanics and compensatory lung growth. In fetal models, phrenic nerve section results in a reduction in both lung growth and alveolar maturation.‘,’ These studies suggest that lung distending pressure (increasing thoracic volume relative to maximum lung volume) and thoracic volume act as regulators of normal postnatal lung growth. Consequently, we speculated that altered mechanical forces produced by phrenectomy or prosthetic replacement of the diaphragm might result in altered thoracic volume relationships, which would in turn, change lung distending pressures and or thoracic volume, resulting in altered regional lung growth. We postulated that there is a relationship between altered mechanical forces and specific changes in regional lung growth, and hypothesized that if this is indeed true, then altering the function of a major muscle of respiration, the diaphragm, should influence regional lung growth. MATERIALS
AND METHODS
Eighteen neonatal piglets (aged 28 days, weighing 7 to 8 kg) were assigned equally to one of three groups: left transthoracic phrenectomy, phrenectomy and diaphragm replacement with a noncompliant silastic patch, or sham thoracotomy. The animals had fluoroscopy before experimentation to document the position of the mediastinum and to note the respiratory excursion of the diaphragm on the involved side. After a mean of 10 days recovery, anesthetized spontaneously breathing piglets were studied supine after preparation with occlusive tracheostomy, placement of esophageal balloon (0.1 mL air inflation) in the midthoracic region, and insertion of specifically designed regional intrapleural pressure monitors (2nd and 8th ICS/MAL) coupled to Validyne pressure transducers (Validyne Engineering Corp, Northridge, CA). Animals were sedated at intervals (every 20 to 25 minutes) with sodium pentobarbital (5 mg/kg) and ketamine (5 to 10 mgikg) sufficient for sedation while enabling spontaneous respirations. Real-time integrated lung volumes were measured by a Hans Rudolf pneumotachograph (Hans Rudolph, Inc, Kansas City, MO) and recorded simultaneously with both esophageal pressure and regional intrapleural pressure on an analog data physiograph. Dynamic compliance (dV/dP) was also calculated from these data. Physiological data were analyzed by an in-line data software acquisition program. The animals were then killed and pressure/volume hysteresis curves were generated by inflating the lungs to a pressure of 30 cm H,O and then extracting sequential 5mL increments of air while recording the pressure at each point.
JournalofPediatric
Surgery, Vol27, No 3 (March), 1992: pp 376-381
377
MECHANICAL FORCES AND NEONATAL LUNG GROWTH
The piglet’s sternum was then opened and the lungs were removed en bloc. They were then divided into four reproducible, anatomic quadrants based on their relationship to the right/left mainstem bronchus (right upper quadrant [RUQ], right lower quadrant [RLQ], left upper quadrant [LUQ], and left lower quadrant [LUQ]. Pulmonary tissue in each quadrant was then assayed for total DNA content (Fleck and Monro technique”‘) and for total protein content (Lowry et al technique”). The results were then averaged for each particular group by quadrant and comparisons were made between the three groups based on these data points. Statistical analysis was carried out via analysis of variance and individual means were tested using Duncan Multiple Range Test.
251
\
Phrenectomy _______Sham ----Patch
RESULTS
Original body weight and weight gain were similar between the three groups. Fluoroscopy showed: (1) sham group: normal diaphragmatic excursion and fixed mediastinum; (2) phrenectomy group: paradoxical motion of an eventrated diaphragm, minimal movement of the mediastinum, and visual loss of thoracic volume on the involved side; and (3) patch group: tethered grossly noncompliant patch with slight paradoxical motion, a fixed mediastinum, and restoration of thoracic volume on the involved side. Pulmona y Function Data Global lung volumes, dynamic compliance (Cdyn) and pressure-volume (P/V) curves as well as regional pleural pressures were reduced in both the phrenectomy and the patch groups compared with the sham controls (Table 1, Fig l), but variations noted among the three groups were not statistically significant. Pleural pressure data (Fig 2) comparing regional differences within the thoracic cavity in each of the study groups showed the following: (1) the sham group exhibited equivalent pleural pressure readings in all four thoracic regions-RUQ(13.107 k 2.95 cm H20), RLQ(13.682 + 3.06 cm HzO), LUQ(13.125 -+ 3.01 cm H,O), LLQ(13.273 k 1.98 cm H,O) (Fig 2); and (2) the phrenectomy group had decreased pleural pressures in the RUQ(11.652 ~fr. 4.11 cm H,O), LUQ(10.460 k 2.87 cm H,O), and LLQ(11.171 -+ 5.61 cm HzO) (Fig 2). The differences among the four regions within this group were not statistically significant. The pleural pressures in the LLQ appeared to be decreased compared with the
0
100
400
200 300 Volume (cc)
Fig 1. Closed chest P/V hysteresis curves generated for the three experimental groups (P = NS).
RLQ but because of the large variability within this group this difference only approached significance. The patch group also exhibited decreased pleural pressures but were limited to the LUQ(ll .OOO+ 4.33 cm HzO) and LLQ(10.344 + 4.61 cm HzO) (Fig 2). The decreases in the LUQ and the LLQ compared with the RUQ(13.423 k 5.38 cm HzO) and RLQ(13.330 + 4.74 cm HzO) were significant (P < .05). The right-sided pressures in the patch group were equivalent to the right-sided pressures found in the sham control. WetlDy Weight, DNAlProtein indexes Total lung wet weights were decreased (P < .05) in the phrenectomy group (74.81 5 7 g) compared with both the sham group (104.23 5 17 g) and the patch group (106.67 + 22 g). Only the phrenectomy group
\_,
---\-. SHAM
PHRENECTOMY
‘A-
PATCH
Table 1. Pulmonary Function Tests
Lung volume
Sham
Phrenectomy
Patch
60.66 f 22
50.06 + 18
42.50 ? 6
Dynamic compliance Right
2.647
3.074
1.949
Left
2.028
2.098
1.919
NOTE. Data given as mean t- SD.
Fig 2. Regional pleural pressures schematically depicted for each experimental group. There was no significant difference in pleural pressure among the sham, phrenectomy, or patch groups. Differences between thoracic regions within each experimental group was significant (“P < .05) for patch LUD < RUQ and LLQ -z RLQ. The patch restored thoracic volume relationships (defect isolated to hemkhorax) compared with the phrenectomy group (three-quadrant effect). Values are expressed in cm H,O.
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PRICE, GALANTOWICZ,
showed significant reductions (P < .OS) in wet weight (Fig 3), dry weight (Fig 4), total DNA content (Fig 5), and total protein content (Fig 6). Neither the sham nor the patch groups showed significant reductions in the above indexes and, in fact, showed consistently similar results. The phrenectomy group showed a significant decrease (P < .05) in total quadrant DNA, total quadrant protein, and wet weights in both the RLQ and LLQ when compared with either sham or patch groups. The phrenectomy group also demonstrated significant reduction (P < .05) in the dry weight of the LLQ(3.43 & 0.94 g) compared with patch and sham groups. Dry weight/wet weight and total protein per gram dry weight and DNA per gram dry weight ratios were not found to be statistically different between the three groups. DISCUSSION
We postulated a relationship between altered mechanical forces and specific changes in regional lung growth hypothesizing that altered function of a major muscle of respiration, the diaphragm, could influence regional lung growth. A previous study of unilateral phrenectomy in cats and piglets” showed unchanged total growth of both lungs combined with an inability to differentiate between decreased growth on the ipsilateral side, increased growth on the contralateral side, or a combination of both. This experiment underscores the importance of evaluating interregional differences in an attempt to isolate specific changes in regional lung growth. By monitoring different regions of the thoracic cavity (regional pleural monitors), we tried to match a specific change in pleural pressure (phrenectomy or nonmobile diaphragm) with a concurrent change in regional lung growth. Phrenectomy causes changes in thoracic configuration, vo1ume,13 and regional intrathoracic pleural pressure.14 The cranial/dorsal pleural pressures increase while the caudalkentral pleural pressures
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Y%ir
Fig 3. Schematic of quadrant wet weights (g) in the three experimental groups. Phrenectomy showed signincant decrease (*P c .Ol) in quadrant wet weights in the RLQ and LLQ compared wlth sham and patch groups.
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7tkii-
Fig 4. Schematic of quadrant dry weights (g) in the three experimental groups. The phrenectomy group showed significant decrease (V c .05) in quadrant dry weights in the LLD compared with the sham and patch groups.
decrease as the scalenes, intercostal, and other accessory muscles of respiration replaced the diaphragm as muscles of respiration. If, as we postulated, lung distending pressure is a component of lung growth, then we would expect the greatest loss of growth to be noted in the area closest to the diaphragm. We would further anticipate diminished growth on the ipsilatera1 side secondary to a loss of thoracic volume. The patch animals with a loss of lung distending pressure (nonmuscular diaphragm), but restitution of thoracic volume should have growth between the two groups. The DNA and total protein data should parallel changes seen in the interregional pleural pressures. Pleural Pressure
These results confirmed differences in interregional transpulmonary pressures within both the patch and phrenectomy group. The phrenectomy group showed a three-quadrant effect with the RUQ joining the LUQ and LLQ in being affected by the left-sided phrenectomy. Also, the LUQ showed a reduction in pleural pressure and not an increase or stabilization as anticipated. The fact that the phrenectomy group did not show statistically significant differences in interregional pleural pressures may be attributed to this three-quadrant affect, although fluoroscopy showed only slight move-
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Fig 5. Schematic of quadrant DNA (mg) in the three experimental groups. Phrenectomy showed significant decrease (*P c .05) in RLQ and LLQ DNA compared with the sham and patch groups.
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Fig 6. Schematic of quadrant protein content (mg) in the three experimental groups. The phrenectomy showed significant decrease (*P c ,051 in RLQ and LLQ protein compared with the sham and patch groups.
ment of the mediastinum in this surgical group. The LLQ pressures were prevented from reaching significance because of the large variability within this one particular group. The patch group also showed a reduction in LUQ and LLQ pleural pressures. This was expected, but of interest is the restoration of the RUQ pleural pressure mimicking that of the right side of the sham animal and isolating the defect to the left side only. The patch animal actually appeared to follow the rules of D’Angelo et all4 in that the cranial segments bilaterally recorded higher pleural pressures than the caudal segments. Biological Assays
The results of the biological assays in the phrenectomy group showed that the quadrants closest to the impaired diaphragm were affected the most and demonstrated a statistically significant reduction in growth compared with the sham animals. Both lower quadrants were affected despite the fact that the pleural pressures on the RLQ were not decreased significantly. It may be that the sensitivity of biological assays is greater than that of the intrapleural monitors. In addition, the relationship between the muscular action of the diaphragm (mechanical forces) and specific changes in regional lung growth, while still valid, raises the possibility that other factors and mediators may be at work. Although the diaphragm is divided by the mediastinum and is supplied by two distinct nerves, impairment of any one half may greatly effect the contralateral side in terms of lung
distending pressure, diaphragmatic excursion, and/or thoracic volume. Whereas these transpulmonary pleural pressures may appear to indicate normal functioning mechanical forces, a more sensitive test (biological assays) shows otherwise. The loss of cell number (DNA) without a change in mean cell size (total protein/DNA) demonstrates a loss of mitotic figures and is reminiscent of cellular hyperplasia and compensatory lung growth.‘” We speculate that forces invoked in compensatory lung growth may be involved in normal postnatal lung growth and that these forces were altered in our study. The patch group showed no reduction in interquadrant wet weight, dry weight, DNA, and total protein when compared with the sham group; however, the pleural pressures were significantly decreased in the affected ipsilateral hemithorax. The mechanics of the patch spared the loss of growth. Because empty thoracic space serves as a stimulus to lung growth (ie, compensatory hyperplasia postpneumonectomy),‘5 a loss of thoracic volume and paradoxical motion of the diaphragm could retard normal lung growth on the effected side. The mechanism for this could involve one or more mechanical forces such as lung distending pressure, thoracic volume, and/or normal diaphragmatic excursion. The presence of a minimally compliant patch in a closed hemithorax undergoing negative pressure during inspiration may generate some level of lung distending pressure. This, coupled with some restoration of thoracic volume and fixed diaphragmatic movement, may be enough to minimize a loss of growth. We speculate that postnatal lung growth is governed in part by parenchymal stretch caused by lung distending pressure, but that an even more important role may be played by either thoracic volume restoration and/or normal diaphragmatic excursion. This may help to explain the loss of growth in the lower quadrants in the phrenectomy group and the apparent normalization of growth in the patch animals despite the fact that lung distending pressure (transpulmonary pressure) was decreased in both. Further study is needed to clearly elucidate the role of these factors in neonatal lung growth.
REFERENCES 1. Dunnill M: Quantitative observations on the anatomy of chronic non-specific lung disease. Med Thorax 22:261-269, 1965 2. Davies G, Reid L: Effect of scoliosis on growth of alveoli and pulmonary arteries and on the right ventricle. Arch Dis Child 46:623-632,197l 3. Cowan MJ, Crystal RG: Lung growth after unilateral pneumonectomy: Quantitation of collagen synthesis and content. Am Rev Respir Dis 111:267-277.1975
4. Simnett JD: Stimulation of cell division following unilateral collapse of the lung. Anat Ret 180:681-686. 1974 5. Arborelius M, Lilja B, Senyk J: Regional and total lung function studies in patients with hemidiaphragmatic paralysis. Respir 321253-264, 1975 6. Ridyard JB, Stewart RM: Regional lung function in unilateral diaphragmatic paralysis. Thorax 31:438-442, 1976 7. Easton PA, Fleetham JA, De La Rocha A, et al: Respiratory
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function after paralysis of the right hemidiaphragm. Am Rev Respir Dis 127:125-128,1983 8. Wigglesworth JS, Desai R: Effects on lung growth of cervical cord section in the rabbit fetus. Ear Hum Dev 3:51-651979 9. Nagai A, Thurlbeck WM, Jansen AH, et al: The effect of chronic biphrenectomy on lung growth and maturation in fetal lambs. Am Rev Respir Dis 137:167-172, 1988 10. Fleck A, Monro HN: The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim Biophys Acta 55:571-583, 1962 11. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275,195l
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12. Mansell AL, Velasquez-Rojas J, Sillos M, et al: Diaphragmatic activity is a determinant of postnatal lung growth. J Appl Physiol61:1098-1103, 1986
13. De Troyer A, Kelly S: Chest wall mechanics in dogs with acute diaphragm paralysis. J Appl Physiol53:373-379,1982 14. D’Angelo E, Sant’Ambrogio G, Agostoni E: Effect of diaphragm activity or paralysis on distribution of pleural pressure. J Appl Physiol37:311-315, 1974 15. Cagle PT, Thurlbeck WM: Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 138:1314-1326,1988
Discussion J.A. Huller (Baltimore, MD): I think the juxtaposi-
tion of these two papers this morning has great pertinence to all of us who care for babies with diaphragmatic hernias. Because I suspect that because we are so pressed in the care of babies who are dying with diaphragmatic hernia and, therefore, carrying out so many clinical trials, we need to come back to this base-what’s going on in the chest. This paper is particularly important because it reminds us that we still have a lot to do in understanding the pathophysiology of the diaphragmatic hernia and understanding better the growth factors related to the lung. The authors have chosen a neonatal subject, the piglet, and I want to focus some of my comments and questions on whether they have yet transferred this to the fetal animal. Because this is where we really need to be looking at the factors associated with not only the development of the diaphragmatic hernia, which remains an enigma, but also which factors result in the abnormal development of the lungs associated with it. There is some good background information and you saw in the initial slide some reference to that neonatal work. But the rapidity of lung growth and the maturation of the pulmonary vascular bed clearly occurs long before there is a piglet, I don’t know what a fetal piglet is called other than that, and it seems to me that focus needs now to be addressed. For example, we know that if a balloon is put in the fetal chest, this will interfere with lung growth and development. Dr Michael Harrison and his associates carried out that study. We had carried out similar studies earlier. We also know from other work that if you ligate the fetal bronchus, the lung does not grow and yet the mechanisms for that remain unclear. Is obstructing drainage from the lung responsible for interfering the changes in the histology and if so how? We need to address the problem itself rather than looking at the clinical manifestations of it. We must continue our clinical trials. But the pessimistic results
from Boston Children’s simply underlines the importance of this paper, which is going back to the laboratory. I would like to ask just a couple of questions and ask the authors if they would project for us some of their thoughts based upon this neonatal model. Do you think it’s possible that the diaphragmatic defect occurs because the lung doesn’t develop? We don’t know the factors responsible for closure of the pleuroperitoneal canal but it may simply be a result of the fact that it isn’t a normal lung to begin with. We know that babies as well as fetal animals who do not have normal pulmonary movement, that is respiratory movement in utero, are often born with major pulmonary abnormalities and indeed other systems may be involved. What is the function of respiratory movement in the developing lung? And does it having something to do with the diaphragm? And, if so, is the development of the diaphragm itself secondary to abnormalities in pulmonary function? I know you have thought about this. Have you also carried out your studies yet in fetal animals? Can you share that with us yet? Or is that going to be on next year’s program? H. F&ton (Knoxville, TN): I’m sort of surprised where Alex focused because my simplistic mind saw this more as a study of eventration and I think what I see is an elegant study showing us why, perhaps, we have to plicate diaphragms. This issue has been debated in this forum in the past and I would just bring it out of the realm of the diaphragmatic hernia and say it looks to me like a very nice model for eventration and tells us a good bit about why sometimes you can’t get babies who have eventration of the diaphragm off ventilators. i&KPrice (response): Thank you Drs Haller and Filston for your comments. Our work involves postnatal rather than fetal lung growth; specifically, growth as it relates to the actions of the respiratory muscles. Fetal and postnatal lung growth are two distinct
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entities and attempts at comparing the two are problematic. We can only speculate on the mediators of fetal lung growth and agree with you that the mechanisms as they pertain to congenital diaphragmatic hernia or any other in utero space occupying thoracic lesion are unclear at this juncture. Certainly, thoracic volume, respiratory movement, and humoral factors all appear to play a substantial role. What we can comment on is that our work suggests that postnatally, particularly in congenital diaphragmatic hernia patients, placement of a noncompliant patch or other attempts to restore thoracic volume may be
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beneficial as an impetus toward improved lung growth on the involved side. Our only reservation about this last statement is that this study was performed on a healthy piglet model and we must be careful when trying to extrapolate animal findings to diseased human lungs. The concept of thoracic volume as a principal mediator of postnatal lung growth is an exciting one, but much work needs to be done in the areas of lung architecture, humoral mediators, and the role of respiratory excursion before this theory can be put into clinical practice.