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Reversible tracheal obstruction in the fetal sheep: Effects on tracheal fluid pressure and lung growth
Reversible Tracheal Obstruction in the Fetal Sheep: Effects on Tracheal Fluid Pressure and Lung Growth By Ezat Hashim, Jean-Martin Laberge, Moy-Fong Chen, and Edmond W. Quillen, Jr Montreal, Quebec • Congenital diaphragmatic hernia (CDH) continues to carry high morbidity and mortality. A number of treatment modalities including extracorporeal membrane oxygenation and in utero repair have improved the mortality rate only minimally. With this condition, there is often insufficient lung mass at birth and persistent pulmonary hypertension postnatally. Experimental fetal tracheal ligation has been shown to increase lung growth in utero and to reduce the hernial contents in CDH. The purpose of this study was to determine the effect of reversible fetal tracheal occlusion on tracheal pressure and lung development. Nine fetal sheep were divided into two groups. Group 1 had intratracheal balloons placed, and the balloons were left inflated for 21 to 28 days. Group 2 consisted of littermates that served as controls. They either had uninflated balloons placed or were left unoperated. Tracheal pressure measurements were recorded periodically, and the amniotic fluid pressure served as a reference. The animals were killed near term, and the lungs, heart, and liver were weighed and corrected for body weight. Standard morphometry was used to compare the lungs further, and the lung DNA and protein content were measured. Tracheal damage from the balloon catheter also was assessed. The tracheal pressure was 3.85 (-+.49 SE) mm Hg in experimental animals, and it averaged -0.27 (-+.27 SE) mm Hg in controls (P < .0001). Tracheal occlusion increased lung weight and volume by two to three times (P < .0001 and P < .0006, respectively) while heart and liver weights remained similar to those of controls. The airspace fraction and radial alveolar counts were increased (P = .044 and P = .0002, respectively), and the alveolar number per body weight was doubled (P < .0001). However, the alveolar number per lung volume was preserved, as were the DNA and protein content per unit weight of lung tissue. The chronic indwelling balloon catheter caused some mucosal and submucosal damage at the balloon site and proximal to it. These results show that tracheal occlusion leads to an elevation of intratracheal pressure that is associated with a tremendous increase in lung growth over a short period in third-trimester fetal sheep. The techniques used in this experiment can be easily modified for use with endoscopic surgical equipment.
ESPITE THE advent of prenatal diagnosis,
extracorporeal membrane oxygenation (ECMO), D and other treatment modalities, congenital diaphrag-
matic hernia (CDH) continues to carry high morbidity and mortality. We have noted clinically that newborns with CDH who undergo prolonged stabilization on a respirator and ECMO have a slow but steady reduction of hernial contents back into the abdomen. In utero, the lungs are fluid-producing organs until a few days before term. The rate of fluid production in fetal sheep has been estimated to be 10 to 14 mL/h or 4.5 mL/kg/h, t,2 Fetuses born with tracheal atresia are found to have lungs that are hyperplastic at both the microscopic and macroscopic levels. 3,4Experimental fetal tracheal ligation has been shown to cause augmented lung growth before birth and reduce the hernia in the CDH animal model, s-9 The effect is believed to be secondary to increased lung fluid retention and elevated tracheal fluid pressures, although the effect on pressure has never been adequately studied. The purposes of this study were to establish a model of reversible tracheal obstruction and to evaluate its effect on tracheal pressure and lung growth in normal fetuses. MATERIALS AND METHODS
From the Department of Surgery, Montreal Children's Hospital, the Departments of Pathology, and Obstetrics & Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec. Presented at the 26th Annual Meeting of the Canadian Association of Paediatric Surgeons, Toronto, Ontario, September 19-21, 1994. Address reprint requests to Jean-Martin Laberge, MD, Department of Surgery, C-1129, Montreal Children's Hospital, 2300 Tupper St, Montreal, Quebec, Canada H3H 1P3. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3008-0016503. 00/0
Time-dated mixed-breed pregnant ewes were operated on at 108 to 118 days' gestation. Rapid induction intubation was performed with thiopental (1 g intrajugularly), and the anesthetic state was maintained with halothane gas. Intravenous access was established and maintained with lactated Ringer's solution. Cefazolin (1 g), gentamicin (150 mg), and Liquamycin (tetracycline; 400 rag) were administered intravenously. After preparation and draping, a lower midtine laparotomy was performed and the uterine horns examined. Once the fetal number and position were established, a standard Swan-Ganz catheter (Baxter Healthcare Corp, Irvine, CA) was tunneled from the left upper flank/lower chest region into the abdominal cavity of the ewe. The fetus was examined by palpation, and the position of the fetal trachea was determined. A small hysterotomy was made over this site, being careful to avoid any cotyledons. Once the amniotic cavity was entered, Babcock clamps were used to tether the edge of the uterine wound to the fetal skin to minimize amniotic fluid spillage and fetal exposure. The catheter was brought into the uterus through a separate site. A tracheotomy was performed by incising the fetal skin, exposing the trachea, and making a small stab wound between two tracheal rings about two fingerbreadths below the larynx. The Swan-Ganz catheter was then inserted into the trachea through this site, up to the level of the catheter thermistor. For experimental animals, catheter balloon was in-
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Journal of Pediatric Surgery, Vo130, No 8 (August), 1995: pp 1172-1177
Copyright © 1 9 9 5 b y W.B, Saunders Company INDEX WORDS: Diaphragmatic hernia, congenital; fetal surgery; fetal lung development; fetal tracheal fluid.
REVERSIBLE TRACHEAL OBSTRUCTION
flated using 0.4 to 0.7 mL of a 1:1 radiocontrast and normal saline mixture. The appropriate balloon inflation was achieved by pulling back intermittently on the catheter during inflation; when resistance was felt, inflation was stopped. Tracheal fluid also stopped leaking at this point. In controls with uninflated balloons, a 6-0 Maxon suture was used to create a snug fit around the catheter to prevent leakage of tracheal fluid. The overlying skin was then reapproximated with a 3-0 silk suture, and the proximal (CVP) port was anchored near the tracheostomy site. The fetus was returned to the uterus, and antibiotics were added to the amniotic fluid. The hysterotomy was closed with either a running 2-0 chromic suture or an automatic stapler, being careful to include all uterine layers in the closure. A 2-0 chromic purse-string suture was used around the catheter's uterine entrance site. The control animals were either untouched twins or those with catheters but with deflated balloons. The catheter was fixed at the ewe exit site with a nonabsorbable suture and was placed in a pouch sutured to the ewe's flank. Postoperatively the ewes received 400 mg of tetracycline intramuscularly each day for 3 days. At weekly postoperative intervals, the ewes were examined with ultrasonography to check for fetal viability. With the ewes quietly standing, the pressure ports from the Swan-Ganz catheters were attached to pressure transducers, and pressure readings were taken at 10 Hz and were averaged over two second blocks for a 30to 40-minute period. Data acquisition was accomplished by driving the pressure signal through a data translation DT2821 analog-todigital converter mounted on an 80486 computer. Because the catheter CVP port reflected the amniotic fluid environment and the distal port reflected the tracheal fluid environment, simultaneous pressure readings of the respective environments could be taken. These readings were stored on disk for later analysis. The data points were then reexamined, and a stable interval of 10 to 30 minutes was selected. The difference between the tracheal and amniotic fluid pressure was calculated and then averaged for each data acquisition session. The averaged readings were eventually grouped, and analysis of variance (ANOVA) was used to compare experimental and control pressure readings. The ewes were kept on the protocol for 3 to 4 weeks until being killed at 137 to 139 days' gestation (full term, 140 to 145 days). A 1-g thiopental injection was followed by a lethal bolus of potassium chloride administered intravenously. Laparotomy and hysterotomy were performed. The umbilical cord was palpated for pulsations, and 250 mg of thiopental was injected into the umbilical vein. To prevent fetal air breathing, the fetus was removed after pulsations had stopped. The catheter was clamped and cut near the tracheostomy site to preserve balloon inflation for later examination. The fetus was then taken, and body weight was measured. The trachea, lungs, and heart were removed as one complex, and the liver was removed separately. The catheter balloon inflation was confirmed by palpation of the trachea before removing it. The trachea was cut 4 cm proximal to the right upper lobe branch point, and the lungs were weighed after carefully dissecting off the heart and esophagus. The liver and ventricular portion of the heart were weighed separately. The right middle lobe and left lingula were tied and cut for snap freezing and were stored at -70°C. These specimens were analyzed later for DNA and protein content, using standard methodsJ °,n The rest of the lung was fixed by instillation of 10% buffered formaldehyde through the trachea at 25-cm hydrostatic pressure for 24 to 48 hours. The upper tracheal segment corresponding to the balloon site was fixed by immersion in the same formaldehyde solution. The fixed lungs were then taken, and lung volumes were measured using the volume displacement method. 12
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The lungs were cut in sagittal sections (3 to 5 mm thick), and random 1- x 1.5-cm blocks were taken from the sections, representing each lobe, and embedded in paraffin. Paraffin blocks were also made of relevant tracheal segments. H&E-stained slides were made from each paraffin block. Lung tissue sections were examined morphometrically using a computerized image analysis system (MCID; Imaging Research Inc, Brock University, St Catherines, Ontario). The system was programmed to measure lung airspace fraction and count alveolar spaces. To simplify the counting process, an alveolar space was counted if it was wholly enclosed by connective tissue or partially enclosed with the remainder of the boundary being the edge of the computer monitor. Fifteen readings were taken for each lung lobe, for a total of 60 readings per lung. The data were transferred to a spreadsheet program, and values for alveolar surface area and alveolar number were calculated using the method of Weibel.m4 Radial alveolar counts (RAC) were taken by hand, using crosshairs incorporated into the microscope objective. ~s-17 A total of 10 readings from each upper and lower lobe were taken by two independent observers at different times, for a total number of 40 readings per animal. Tracheal segments were examined simultaneously by two observers for any pathological changes. To allow for comparison between experimental and control groups, data were corrected for fetal body weight where appropriate. Statistical analysis was performed using the Student's t test, or ANOVA when appropriate. A Scheffe test was applied for confirmation when a small amount of data were obtained. Values for means were expressed in terms of standard error. P values were considered significant when less than .05. RESULTS
T h e first five e w e s t h a t w e r e o p e r a t e d o n a b o r t e d b e f o r e t e r m . M o s t o f t h e s e a n i m a l s m i s c a r r i e d 10 days postoperatively, with fetuses that were dead and s h o w e d e v i d e n c e o f s e v e r e autolysis a n d i n f e c t i o n . T h e p r o t o c o l w a s m o d i f i e d to i n c l u d e t e t r a c y c l i n e ( f o r c h l a m y d i a ) , to c a n n u l a t e o n l y a s i n g l e f e t u s p e r e w e , a n d to e x p o s e as little o f t h e f e t u s as p o s s i b l e d u r i n g t h e p r o c e d u r e . T h e p r o t o c o l w a s finally as p r e s e n t e d in t h e M a t e r i a l s a n d M e t h o d s . H a v i n g d o n e this, t h e s u b s e q u e n t f o u r e w e s w e n t s u c c e s s f u l l y to t e r m . B e c a u s e o f t w i n a n d t r i p l e t g e s t a t i o n s , t h e r e w e r e f o u r e x p e r i m e n t a l a n d five control fetuses.
Pressure Measurements T h e m e a n t r a c h e a l p r e s s u r e w a s 3.85 +- .49 m m H g f o r e x p e r i m e n t a l a n i m a l s a n d - 0 . 2 7 + .27 m m H g f o r c o n t r o l a n i m a l s . T h e d i f f e r e n c e w a s statistically signific a n t ( P < .0001).
Gross Parameters A f t e r d e a t h , t h e e x p e r i m e n t a l l u n g s w e r e f o u n d to be much larger than those of control specimens, with tissue growth bulging through intercostal spaces and c o s t o d i a p h r a g m a t i c r e c e s s e s . T h e tissue a p p e a r e d m o r e t r a n s l u c e n t a n d boggy. T h e c a t h e t e r p o r t s w e r e c h e c k e d a n d f o u n d to b e in g o o d l o c a t i o n in all cases.
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HASHIM ET AL
Fig 1. Comparison between experimental (left) and control (right) lungs. The specimens include the heart as a point of reference for size. Note the slight dilatation of the lower trachea in the experimental animal (balloon site).
A small dilatation of the trachea was usually present at the balloon site (Fig 1). Fetal lung, liver, and heart weight were compared, and the only significant difference was with respect to the lungs (Table 1). Postfixation lung volumes also were significantly different. Pulmonary parameters for experimental animals were two to three times those of the controls with respect to weight and volume. Among the controls, there was no difference between the animals left untouched and those with an uninflated balloon catheter in place.
Morphometric Measurements Histological examination showed a slightly greater alveolar number and thinner septations in the experimental lungs; otherwise the architecture appeared normal when compared with that of control specimens. The arteries appeared normal, and capillaries containing red blood cells were noted in the alveolar septae. The image analyzer was programmed to measure airspace fraction and count the number of alveoli per Table 1. Summary of Gross Measurements on Different Organ Systems Measurement LW/BW (%) LV/BW (mL/g) HW/BW (%) Li/BW (%)
Experimental 6.76 .15 .478 2.29
-+ .43 -+ .017 -+ .027 _+ 0.31
Control 2.62 .058 .468 1.88
_+ .20 -+ .005 -+ .021 -4- 0.14
unit area. The airspace fraction was found to be greater in experimental animals (Table 2). We found that the alveolar numerical density did not vary statistically between the two groups. However, because the size of the lungs differed between the control and experimental groups, a calculation of alveolar number per unit weight of fetus showed a doubling of the number of alveoli in experimental animals (Table 2). This supports the notion that the lung tissue that develops as a result of the tracheal occlusion does so while the ratio between parenchyma and airspace volume is preserved. The total alveolar surface area was derived and normalized for fetal body weight (Table 2). This result showed highly significant differences between the two groups, with the experimental animal lungs having more than twice the surface area of control lungs. The values for the radial alveolar count were different between the two groups, indicating that the number of gas exchanging units distal to the terminal bronchiole was higher in the experimental animals. Table 2. Summary of Morphometric Measurements of Lung Tissue Measurement
P Value < .0001 .0006 .78 .24
Abbreviations: LW, lung weight; BW, body weight; LV, lung volume; HW, heart weight; Li, liver weight.
Airspace fraction AIv/BW (no. x 106/g) AIv/LV (no. x 106/mL) SA/BW (cm2/g) RAC
Experimental (n = 4) ,72 1.10 7.31 110.8 7.86
+ .03 -+ .040 -+ .81 -+ 10.8 -+ .34
Control (n = 5) .64 .536 9.37 43.4 5.33
+ .02 -+ .049 -+ .92 _+ 2.9 + .19
P Value .044 <.0001 .14 .0003 .0002
Abbreviations: AIv, total alveolar number; BW, fetal body weight; LV, total lung volume; SA, total alveolar surface area; RAC, radial alveolar count.
REVERSIBLE TRACHEAL OBSTRUCTION
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DNA and Protein Analysis No statistically significant differences were found in the analysis of DNA or protein levels per unit weight of lung tissue. The ratio of protein to DNA levels was preserved between the two groups, !mplying that lung growth had occurred through cell multiplication and not through hypertrophy of existing lung elements. When examining thes.e data in terms of fetal body weight, the amounts 0f lung DNA and protein are found to be more than doubled in experimental animals. The results are summarized in Table 3.
Tracheal Histology Changes noted in cannulated control animals inelude flattening of the mucosa just below the tracheostomy site, with fibrotie changes and thinning of the submueosa attributed to intratracheal rubbing by the Swan-Ganz catheter. In experimental animals, squamous metaplasia was apparent at the balloon site, with areas of epithelial necrosis and sloughing. Submucosal changes ranged from granulation tissue infiltration, and polymorphonueleocge Or lymphocyte infiltration, to focal areas of fibrosis and calcification. Changes below the balloon site either were not apparent or were minimal with areas of mucosal thinning. The effect on the tracheal cartilage was minimal, being restricted to perichondrial thickening and increased separation of the posterior free ends of the cartilage. However, at this posterior end, the trachealis muscle was found to be thickened at and below the balloon site. At the tracheostomy site, a squamous epithelial tract could be seen connecting the skin to the tracheal mucosa. DISCUSSION
The effect of balloon occlusion of the fetal trachea appears comparable to the effect of laryngeal atresia and tracheal ligation in terms of lung morphology and morphometry. The present study is the first to show the in vivo changes in tracheal pressure associated with tracheal obstruction. Table 3. Summary of DNA and Protein Analysis of Lung Tissue Measurement Protein (mg/g lung) DNA (mg/g lung) protein/DNA Protein]BW (mg/g) DNA/BW (mg/g)
Experimental 26 2.8 10.0 1.68 0.186
-+ 7 + .2 + 3,6 + .41 +- .017
Control 20 2.8 7.3 0.52 0.073
+ 4 _+ .2 _+ 1.2 +_ .11 _+ .009
P Value .48 .97 .45 .019 .0004
NOTE.'The first two values were derived per ,gram of wet weight of lung. The last two values were derived in terms of fetal body weight (BW)~
The tracheal pressure measurements in experimental animals were found to be in the range of 4 mm Hg, while those of control animals fluctuated around zero, relative to amniotic fluid pressure. Other groups reported control values of 1.25 to 3.0 mm Hg relative to amniotic fluid pressures. 18-2° However, in most of these studies the relative placement of the tracheal and amniotic fluid catheters is unclear. 19,2° In one study the amniotic fluid pressure was derived from the difference between the maternal intraabdominal pressure and the intrauterine pressure, is In contrast, the present study maintained a controlled relative distance between the two catheters by fixing the amniotic fluid pressure port near the fetal tracheos, tomy site. The difference in pressure between the two ports is what was calculated as the tracheal fluid pressure. Values from experimental animals show that tracheal obstruction is indeed related to an elevation in tracheal fluid pressure by a magnitude of 4 mm Hg. DiFiore et al obtain pressures of 0 mm Hg for control animals and 6 mm Hg for experimenta ! tracheally ligated animals. 8 However, in their study, pressures were measured only at the time of fetal delivery and were relative to air. The present in vivo preparation is probably a better reflection of the true changes. The increase in intratracheal pressure associated with tracheal obstruction is not surprising. As was mentioned earlier, the fetal lungs act as fluidsecreting organs up until near-term, and thereby contribute to the amniotic ~gid volume, zl-25 Active transport across the pulmQna~ epithelium eventually diminishes near term, in coordination with rising fetal cortisol levels, preparing the lungs for air-breathing. 26 Thus, it is expected that tracheal ligation or obstruction would result in fluid secretion in an enclosed space and would increase the intratracheal pressure. The pressure increase would continue until a plateau is reached 4 mm Hg in the present study. What is surprising is that such a small change in pressure is associated with a large difference in overall lung development. The gross changes associated with tracheal occlusion are comparable to those seen in other studies in terms of lung growth and control values. 5,8,9,2a,27,28 Lung weight and volume are increased by a factor of 2 to 3 with both tracheal obstruction and tracheal ligation. These lungs are obviously large for gestational age and appear normal histologically. As was shown in the present study, the effect of organ growth appears to be confined to the lung, because the heart and liver are not affected. This is consistent with what one sees in human congenital laryngeal atresia?
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Histological and morphometric analyses were used to compare lung architecture. From this it is evident that lung growth in response to tracheal obstruction results in an increased number of lung units--not simply an increase in the size of preexisting units. The airspace fraction was slightly higher in experimental lungs, and the alveolar wall was thinner, characteristics found in more mature lungs. 29 The alveolar surface area and alveolar number did not differ significantly between the two groups when expressed per unit of lung weight or volume. However, when expressed in terms of fetal body weight, the obstructed lungs show a two- to threefold increase in these values relative to those of controls. These changes are essentially proportional to those occurring at the macroscopic level. Most values obtained were consistent with those of other studies, 8,9 the exception being with alveolar number, probably a reflection of different methods in counting alveoli. The radial alveolar count (RAC) was found to be significantly different between obstructed and control lungs. This shows that alveolar complexity beyond the terminal bronchiole increases with this intervention.15.~6 To assess whether the tissue growth is related hyperplasia or hypertrophy, tissue DNA and protein content may be used. 8,9,24,3°,31 Increased mitosis will lead to an increase in tissue DNA levels. Increased production of interstitial matrix and cytoplasm will lead to elevated tissue protein levels. The ratio of tissue DNA to protein levels would be expected to remain constant if the balance between the number of ceils and the amount of extracellular matrix remains constant. In this study, both tissue DNA and protein levels between experimental and control lungs were unchanged when expressed per gram of lung tissue. The ratio of DNA to protein also was unchanged. However, because the experimental lungs were much larger, these values in terms of fetal body weight were significantly different, indicating that cell number and quantity of interstitium are elevated proportionally after tracheal obstruction. Analyses of the changes to the trachea show that damage occurs along the tracheal segment, between the tracheostomy site and the Swan-Ganz catheter balloon. The mucosa adjacent to the catheter tubing exhibits squamous metaplasia, probably from chronic rubbing by the catheter against the mucosa. At the balloon site the damage was moresevere, ranging from squamous metaplasia to frank necrosis and sloughing. It is comforting to note that even with the crude obturator used in this study, these changes were confined to the mucosa and submucosa, while
HASHIM ET AL
the cartilage was left relatively unaffected. Nevertheless it would be worthwhile to design a clinically applicable obturator that would minimize tracheal damage. It is evident from this study and others that prevention of fluid egress from the potential airspaces in the developing fetus results in accelerated lung growth.5,7-9,21 One proposed mechanism of action is the modulation of local growth factors via airspace pressure receptors. 8 An increase in lung fluid pressure may augment a local growth cascade. The effect on growth has been shown to confine itself strictly to areas of lung fluid retention, indicating the importance of fluid retention in this mechanism. 24 Systemically circulating growth factors would probably be ineffective without tracheal obstruction. Whereas pressure tends to cause necrosis of tissue (as is frequently seen with abcesses and tumor growth), tension can increase the mitotic activity in various tissues. Epithelial cell division in response to tension has been examined by several investigators. Increased mechanical stress applied to cultured fibroblasts was shown to increase the mitotic activity by speeding up the cell cycle. 32 Others observed the effect of applied tension in vivo on mouse skin and confirm increased mitotic activity in response to this intervention.33 Liu et al studied the effect of mechanical stretch on proliferation of fetal rat lung cells2 4 They concluded that mechanical forces act directly to stimulate fetal rat lung cell growth and that this effect is not mediated by prostaglandins or leukotrienes. The concept of tension affecting tissue growth has been applied to clinical practice for several years with the use of skin expanders. With fetal surgery becoming a reality in the clinical setting, methods of minimizing the surgical complications are being investigated. One of the gravest complications is preterm labor, which is often difficult to control medically. The uterine instability is believed to be secondary to the hysterotomy wound. 35 Thus, it seems logical that reducing the size of the wound may decrease this dreaded complication. Modern technology has allowed minimally invasive surgery with laparoscopic equipment, and this has recently been used in the experimental setting in fetal s u r g e r y . 3639 Our tracheal occlusion technique could easily be modified to incorporate the use of these techniques. ACKNOWLEDGMENT The authors are thankful to lain Vowels for his expert technical assistance in the animal care and experiments, and to Minh Duong for performing the DNA and protein assays.
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1. Adamson TM, Brodecky V, Lambert TF, et al: Lung liquid production and composition in the "in utero" foetal lamb. AJEBAK 53:65-75, 1975 2. Harding R, Sigger JN, Wickham PJ, et al: The regulation of flow of pulmonary fluid in fetal sheep. Resp Physiol 57:47-59, 1984 3. Wigglesworth JS, Desai R, Hislop AA: Fetal lung growth in congenital laryngeal atresia. Pediatr Pathol 7:515-525, 1987 4. Richards DS, Yancey MK, Duff P, et al: The perinatal management of severe laryngeal stenosis. Obstet Gynecol 80:537540, 1992 5. Alcorn D, Adamson TM, Lambert TF, et al: Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649-660, 1977 6. Lanman JT, Schaffer A, Herod L, et al: Distensibility of the fetal lung with fluid in sheep. Pediatr Res 5:586-590, 1971 7. Carmel AJ, Friedman F, Adams FH: Fetal tracheal ligation and lung development. Am .l Dis Child 109:452-456, 1965 8. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg 29:248-257, 1994 9. Wilson JM, DiFiore JW, Peters CA: Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1440. 1993 10. Downs TR, Wilfinger WW: Fluorometric quantification of DNA in cells and tissue. Anal Biochem 131:538-547, 1983 11. Lowry OH. Rosebrough NJ, Farr AL, et al: Protein measurements with the folin phenol reagent. J Biol Chem 193:265-275.1951 12. Scherle W: A simple method for volumetry of organs in quantitative stereology. Mikroskopie 26:57-60. 1970 13. Weibel ER. Gomez DM: A principle for counting tissue structures on random sections. J Appl Physiol 17:343-348. 1962 14. Weibel ER: A simplified morphometric method for estimating diffusing capacity in normal and emphysematous human lungs. Am Rev Respir Dis 107:579-588, 1973 15. Cooney TP, Thurlbeck WM: The radial alveolar count method of Emery and Mitbal: A reappraisal 2 Intrauterine and early postnatal lung growth. Thorax 37:580-583, 1982 16. Cooney TP, Thurlbeck WM: The radial alveolar count method of Emery and Mithal: A reappraisal 1--Postnatal lung growth. Thorax 37:572-579. 1982 17. Nakamura Y, Harada K, Yamamoto I. et al: Human pulmonary hypoplasia. Statistical. morphological, morphometric, and biochemical study. Arch Pathol Lab Med 116:635-642. 1992 18. Harding R, Hooper SB. Dickson KA: A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydramnios. Am J Obstet Gynecol 163:1904-1913. 1990 19. Vilos GA. Liggins GC: Intrathoracic pressures in fetal sheep. J Dev Physiol 4:247-256, 1982 20. Fewell JE0 Johnson P: Upper airway dynamics during breathing and during apnoea in fetal lambs. J Physiol 339:495-504. 1983 21. Adzick NS, Harrison MR, Glick PL, et al: Experimental pulmonary hypoplasia and oligohydramnios: Relative contribu-
tions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-665, 1984 22. Benson BJ, Kitterman JA, Clements JA, et al: Changes in phospholipid composition of lung surfactant during development in the fetal lamb. Biochim Biophys Acta 753:83-88, 1983 23. Fewell JE, Hislop AA, Kitterman JA, et al: Effect of tracheostomy on lung development in fetal lambs. J Appl Physiol 55:1103-1108, 1983 24. Moessinger AC, Harding R, Adamson TM, et al: Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86:1270-1277, 1990 25. Rethmeier HB, Egberts H: Transversal and longitudinal lecithin-sphingomyelin ratios in amniotic and lung fluids of fetal lambs. Am J Obstet Gynecol 122:593-596, 1975 26. Kitterman JA, Ballard PL, Clements JA, et al: Tracheal fluid in fetal lambs: Spontaneous decrease prior to birth. J Appl Physiol 47:985-989, 1979 27. Adzick NS, Outwate r KM, Harrison MR, et al: Correction of congenital diaphragmatic hernia in utero. IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 28. Kitterman JA: Fetal lung development. J Dev Physiol 6:67-82, 1984 (review) 29. Docimo SG, Crone RK, Davies P, et al: Pulmonary development in the fetal lamb: Morphometric study of the alveolar phase. Anat Rec 229:495-498, 1991 30. Wigglesworth JS, Desai R: Use of DNA estimation for growth assessment in normal and hypoplastic fetal lungs. Arch Dis Child 56:601-605, 1981 31. Hosoda Y, Rossman JE, Glick PL: Pathophysiology of congenital diaphragmatic hernia. IV: Renal hyperplasia is associated with pulmonary hypoplasia. J Pediatr Surg 28:464-469, 1993 32. curtis AS, Seehar GM: The control of cell division by tension or diffusion. Nature 274:52-53, 1978 33. Squier CA: The stretching of mouse skin in vivo: Effect on epidermal proliferation and thickness. J Invest Dermatol 74:68-71, 1980 34. Liu M, Skinner SJ, Xu J, et al: Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol 263:376-383, 1992 35. Harrison MR, Golbus MS, Filly RA: The Unborn Patient: Prenatal Diagnosis and Treatment, chap 28. Philadelphia, PA, Saunders, 1991 36. Luks FI, Deprest J, Marcus M, et al: Carbon dioxide pneumoamnios causes acidosis in fetal lamb. Fetal Diagn Ther 9:105-109, 1994 37. Luks FI, Deprest JA, Vandenberghe K, et al: A model for fetal surgery through intrauterine endoscopy. J Pediatr Surg 29:1007-1009, 1994 38. Estes JM, Whitby D J, Lorenz HP, et al: Endoscopic creation and repair of fetal cleft lip. Plast Reconstr Surg 90:743-746, 1992 39. Estes JM, MacGillivray TE, Hedrick MH, et al: Fetoscopic surgery for the treatment of congenital anomalies. J Pediatr Surg 27:950-954, 1992
ESPITE THE advent of prenatal diagnosis,
extracorporeal membrane oxygenation (ECMO), D and other treatment modalities, congenital diaphrag-
matic hernia (CDH) continues to carry high morbidity and mortality. We have noted clinically that newborns with CDH who undergo prolonged stabilization on a respirator and ECMO have a slow but steady reduction of hernial contents back into the abdomen. In utero, the lungs are fluid-producing organs until a few days before term. The rate of fluid production in fetal sheep has been estimated to be 10 to 14 mL/h or 4.5 mL/kg/h, t,2 Fetuses born with tracheal atresia are found to have lungs that are hyperplastic at both the microscopic and macroscopic levels. 3,4Experimental fetal tracheal ligation has been shown to cause augmented lung growth before birth and reduce the hernia in the CDH animal model, s-9 The effect is believed to be secondary to increased lung fluid retention and elevated tracheal fluid pressures, although the effect on pressure has never been adequately studied. The purposes of this study were to establish a model of reversible tracheal obstruction and to evaluate its effect on tracheal pressure and lung growth in normal fetuses. MATERIALS AND METHODS
From the Department of Surgery, Montreal Children's Hospital, the Departments of Pathology, and Obstetrics & Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec. Presented at the 26th Annual Meeting of the Canadian Association of Paediatric Surgeons, Toronto, Ontario, September 19-21, 1994. Address reprint requests to Jean-Martin Laberge, MD, Department of Surgery, C-1129, Montreal Children's Hospital, 2300 Tupper St, Montreal, Quebec, Canada H3H 1P3. Copyright © 1995 by W.B. Saunders Company 0022-3468/95/3008-0016503. 00/0
Time-dated mixed-breed pregnant ewes were operated on at 108 to 118 days' gestation. Rapid induction intubation was performed with thiopental (1 g intrajugularly), and the anesthetic state was maintained with halothane gas. Intravenous access was established and maintained with lactated Ringer's solution. Cefazolin (1 g), gentamicin (150 mg), and Liquamycin (tetracycline; 400 rag) were administered intravenously. After preparation and draping, a lower midtine laparotomy was performed and the uterine horns examined. Once the fetal number and position were established, a standard Swan-Ganz catheter (Baxter Healthcare Corp, Irvine, CA) was tunneled from the left upper flank/lower chest region into the abdominal cavity of the ewe. The fetus was examined by palpation, and the position of the fetal trachea was determined. A small hysterotomy was made over this site, being careful to avoid any cotyledons. Once the amniotic cavity was entered, Babcock clamps were used to tether the edge of the uterine wound to the fetal skin to minimize amniotic fluid spillage and fetal exposure. The catheter was brought into the uterus through a separate site. A tracheotomy was performed by incising the fetal skin, exposing the trachea, and making a small stab wound between two tracheal rings about two fingerbreadths below the larynx. The Swan-Ganz catheter was then inserted into the trachea through this site, up to the level of the catheter thermistor. For experimental animals, catheter balloon was in-
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Journal of Pediatric Surgery, Vo130, No 8 (August), 1995: pp 1172-1177
Copyright © 1 9 9 5 b y W.B, Saunders Company INDEX WORDS: Diaphragmatic hernia, congenital; fetal surgery; fetal lung development; fetal tracheal fluid.
REVERSIBLE TRACHEAL OBSTRUCTION
flated using 0.4 to 0.7 mL of a 1:1 radiocontrast and normal saline mixture. The appropriate balloon inflation was achieved by pulling back intermittently on the catheter during inflation; when resistance was felt, inflation was stopped. Tracheal fluid also stopped leaking at this point. In controls with uninflated balloons, a 6-0 Maxon suture was used to create a snug fit around the catheter to prevent leakage of tracheal fluid. The overlying skin was then reapproximated with a 3-0 silk suture, and the proximal (CVP) port was anchored near the tracheostomy site. The fetus was returned to the uterus, and antibiotics were added to the amniotic fluid. The hysterotomy was closed with either a running 2-0 chromic suture or an automatic stapler, being careful to include all uterine layers in the closure. A 2-0 chromic purse-string suture was used around the catheter's uterine entrance site. The control animals were either untouched twins or those with catheters but with deflated balloons. The catheter was fixed at the ewe exit site with a nonabsorbable suture and was placed in a pouch sutured to the ewe's flank. Postoperatively the ewes received 400 mg of tetracycline intramuscularly each day for 3 days. At weekly postoperative intervals, the ewes were examined with ultrasonography to check for fetal viability. With the ewes quietly standing, the pressure ports from the Swan-Ganz catheters were attached to pressure transducers, and pressure readings were taken at 10 Hz and were averaged over two second blocks for a 30to 40-minute period. Data acquisition was accomplished by driving the pressure signal through a data translation DT2821 analog-todigital converter mounted on an 80486 computer. Because the catheter CVP port reflected the amniotic fluid environment and the distal port reflected the tracheal fluid environment, simultaneous pressure readings of the respective environments could be taken. These readings were stored on disk for later analysis. The data points were then reexamined, and a stable interval of 10 to 30 minutes was selected. The difference between the tracheal and amniotic fluid pressure was calculated and then averaged for each data acquisition session. The averaged readings were eventually grouped, and analysis of variance (ANOVA) was used to compare experimental and control pressure readings. The ewes were kept on the protocol for 3 to 4 weeks until being killed at 137 to 139 days' gestation (full term, 140 to 145 days). A 1-g thiopental injection was followed by a lethal bolus of potassium chloride administered intravenously. Laparotomy and hysterotomy were performed. The umbilical cord was palpated for pulsations, and 250 mg of thiopental was injected into the umbilical vein. To prevent fetal air breathing, the fetus was removed after pulsations had stopped. The catheter was clamped and cut near the tracheostomy site to preserve balloon inflation for later examination. The fetus was then taken, and body weight was measured. The trachea, lungs, and heart were removed as one complex, and the liver was removed separately. The catheter balloon inflation was confirmed by palpation of the trachea before removing it. The trachea was cut 4 cm proximal to the right upper lobe branch point, and the lungs were weighed after carefully dissecting off the heart and esophagus. The liver and ventricular portion of the heart were weighed separately. The right middle lobe and left lingula were tied and cut for snap freezing and were stored at -70°C. These specimens were analyzed later for DNA and protein content, using standard methodsJ °,n The rest of the lung was fixed by instillation of 10% buffered formaldehyde through the trachea at 25-cm hydrostatic pressure for 24 to 48 hours. The upper tracheal segment corresponding to the balloon site was fixed by immersion in the same formaldehyde solution. The fixed lungs were then taken, and lung volumes were measured using the volume displacement method. 12
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The lungs were cut in sagittal sections (3 to 5 mm thick), and random 1- x 1.5-cm blocks were taken from the sections, representing each lobe, and embedded in paraffin. Paraffin blocks were also made of relevant tracheal segments. H&E-stained slides were made from each paraffin block. Lung tissue sections were examined morphometrically using a computerized image analysis system (MCID; Imaging Research Inc, Brock University, St Catherines, Ontario). The system was programmed to measure lung airspace fraction and count alveolar spaces. To simplify the counting process, an alveolar space was counted if it was wholly enclosed by connective tissue or partially enclosed with the remainder of the boundary being the edge of the computer monitor. Fifteen readings were taken for each lung lobe, for a total of 60 readings per lung. The data were transferred to a spreadsheet program, and values for alveolar surface area and alveolar number were calculated using the method of Weibel.m4 Radial alveolar counts (RAC) were taken by hand, using crosshairs incorporated into the microscope objective. ~s-17 A total of 10 readings from each upper and lower lobe were taken by two independent observers at different times, for a total number of 40 readings per animal. Tracheal segments were examined simultaneously by two observers for any pathological changes. To allow for comparison between experimental and control groups, data were corrected for fetal body weight where appropriate. Statistical analysis was performed using the Student's t test, or ANOVA when appropriate. A Scheffe test was applied for confirmation when a small amount of data were obtained. Values for means were expressed in terms of standard error. P values were considered significant when less than .05. RESULTS
T h e first five e w e s t h a t w e r e o p e r a t e d o n a b o r t e d b e f o r e t e r m . M o s t o f t h e s e a n i m a l s m i s c a r r i e d 10 days postoperatively, with fetuses that were dead and s h o w e d e v i d e n c e o f s e v e r e autolysis a n d i n f e c t i o n . T h e p r o t o c o l w a s m o d i f i e d to i n c l u d e t e t r a c y c l i n e ( f o r c h l a m y d i a ) , to c a n n u l a t e o n l y a s i n g l e f e t u s p e r e w e , a n d to e x p o s e as little o f t h e f e t u s as p o s s i b l e d u r i n g t h e p r o c e d u r e . T h e p r o t o c o l w a s finally as p r e s e n t e d in t h e M a t e r i a l s a n d M e t h o d s . H a v i n g d o n e this, t h e s u b s e q u e n t f o u r e w e s w e n t s u c c e s s f u l l y to t e r m . B e c a u s e o f t w i n a n d t r i p l e t g e s t a t i o n s , t h e r e w e r e f o u r e x p e r i m e n t a l a n d five control fetuses.
Pressure Measurements T h e m e a n t r a c h e a l p r e s s u r e w a s 3.85 +- .49 m m H g f o r e x p e r i m e n t a l a n i m a l s a n d - 0 . 2 7 + .27 m m H g f o r c o n t r o l a n i m a l s . T h e d i f f e r e n c e w a s statistically signific a n t ( P < .0001).
Gross Parameters A f t e r d e a t h , t h e e x p e r i m e n t a l l u n g s w e r e f o u n d to be much larger than those of control specimens, with tissue growth bulging through intercostal spaces and c o s t o d i a p h r a g m a t i c r e c e s s e s . T h e tissue a p p e a r e d m o r e t r a n s l u c e n t a n d boggy. T h e c a t h e t e r p o r t s w e r e c h e c k e d a n d f o u n d to b e in g o o d l o c a t i o n in all cases.
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HASHIM ET AL
Fig 1. Comparison between experimental (left) and control (right) lungs. The specimens include the heart as a point of reference for size. Note the slight dilatation of the lower trachea in the experimental animal (balloon site).
A small dilatation of the trachea was usually present at the balloon site (Fig 1). Fetal lung, liver, and heart weight were compared, and the only significant difference was with respect to the lungs (Table 1). Postfixation lung volumes also were significantly different. Pulmonary parameters for experimental animals were two to three times those of the controls with respect to weight and volume. Among the controls, there was no difference between the animals left untouched and those with an uninflated balloon catheter in place.
Morphometric Measurements Histological examination showed a slightly greater alveolar number and thinner septations in the experimental lungs; otherwise the architecture appeared normal when compared with that of control specimens. The arteries appeared normal, and capillaries containing red blood cells were noted in the alveolar septae. The image analyzer was programmed to measure airspace fraction and count the number of alveoli per Table 1. Summary of Gross Measurements on Different Organ Systems Measurement LW/BW (%) LV/BW (mL/g) HW/BW (%) Li/BW (%)
Experimental 6.76 .15 .478 2.29
-+ .43 -+ .017 -+ .027 _+ 0.31
Control 2.62 .058 .468 1.88
_+ .20 -+ .005 -+ .021 -4- 0.14
unit area. The airspace fraction was found to be greater in experimental animals (Table 2). We found that the alveolar numerical density did not vary statistically between the two groups. However, because the size of the lungs differed between the control and experimental groups, a calculation of alveolar number per unit weight of fetus showed a doubling of the number of alveoli in experimental animals (Table 2). This supports the notion that the lung tissue that develops as a result of the tracheal occlusion does so while the ratio between parenchyma and airspace volume is preserved. The total alveolar surface area was derived and normalized for fetal body weight (Table 2). This result showed highly significant differences between the two groups, with the experimental animal lungs having more than twice the surface area of control lungs. The values for the radial alveolar count were different between the two groups, indicating that the number of gas exchanging units distal to the terminal bronchiole was higher in the experimental animals. Table 2. Summary of Morphometric Measurements of Lung Tissue Measurement
P Value < .0001 .0006 .78 .24
Abbreviations: LW, lung weight; BW, body weight; LV, lung volume; HW, heart weight; Li, liver weight.
Airspace fraction AIv/BW (no. x 106/g) AIv/LV (no. x 106/mL) SA/BW (cm2/g) RAC
Experimental (n = 4) ,72 1.10 7.31 110.8 7.86
+ .03 -+ .040 -+ .81 -+ 10.8 -+ .34
Control (n = 5) .64 .536 9.37 43.4 5.33
+ .02 -+ .049 -+ .92 _+ 2.9 + .19
P Value .044 <.0001 .14 .0003 .0002
Abbreviations: AIv, total alveolar number; BW, fetal body weight; LV, total lung volume; SA, total alveolar surface area; RAC, radial alveolar count.
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DNA and Protein Analysis No statistically significant differences were found in the analysis of DNA or protein levels per unit weight of lung tissue. The ratio of protein to DNA levels was preserved between the two groups, !mplying that lung growth had occurred through cell multiplication and not through hypertrophy of existing lung elements. When examining thes.e data in terms of fetal body weight, the amounts 0f lung DNA and protein are found to be more than doubled in experimental animals. The results are summarized in Table 3.
Tracheal Histology Changes noted in cannulated control animals inelude flattening of the mucosa just below the tracheostomy site, with fibrotie changes and thinning of the submueosa attributed to intratracheal rubbing by the Swan-Ganz catheter. In experimental animals, squamous metaplasia was apparent at the balloon site, with areas of epithelial necrosis and sloughing. Submucosal changes ranged from granulation tissue infiltration, and polymorphonueleocge Or lymphocyte infiltration, to focal areas of fibrosis and calcification. Changes below the balloon site either were not apparent or were minimal with areas of mucosal thinning. The effect on the tracheal cartilage was minimal, being restricted to perichondrial thickening and increased separation of the posterior free ends of the cartilage. However, at this posterior end, the trachealis muscle was found to be thickened at and below the balloon site. At the tracheostomy site, a squamous epithelial tract could be seen connecting the skin to the tracheal mucosa. DISCUSSION
The effect of balloon occlusion of the fetal trachea appears comparable to the effect of laryngeal atresia and tracheal ligation in terms of lung morphology and morphometry. The present study is the first to show the in vivo changes in tracheal pressure associated with tracheal obstruction. Table 3. Summary of DNA and Protein Analysis of Lung Tissue Measurement Protein (mg/g lung) DNA (mg/g lung) protein/DNA Protein]BW (mg/g) DNA/BW (mg/g)
Experimental 26 2.8 10.0 1.68 0.186
-+ 7 + .2 + 3,6 + .41 +- .017
Control 20 2.8 7.3 0.52 0.073
+ 4 _+ .2 _+ 1.2 +_ .11 _+ .009
P Value .48 .97 .45 .019 .0004
NOTE.'The first two values were derived per ,gram of wet weight of lung. The last two values were derived in terms of fetal body weight (BW)~
The tracheal pressure measurements in experimental animals were found to be in the range of 4 mm Hg, while those of control animals fluctuated around zero, relative to amniotic fluid pressure. Other groups reported control values of 1.25 to 3.0 mm Hg relative to amniotic fluid pressures. 18-2° However, in most of these studies the relative placement of the tracheal and amniotic fluid catheters is unclear. 19,2° In one study the amniotic fluid pressure was derived from the difference between the maternal intraabdominal pressure and the intrauterine pressure, is In contrast, the present study maintained a controlled relative distance between the two catheters by fixing the amniotic fluid pressure port near the fetal tracheos, tomy site. The difference in pressure between the two ports is what was calculated as the tracheal fluid pressure. Values from experimental animals show that tracheal obstruction is indeed related to an elevation in tracheal fluid pressure by a magnitude of 4 mm Hg. DiFiore et al obtain pressures of 0 mm Hg for control animals and 6 mm Hg for experimenta ! tracheally ligated animals. 8 However, in their study, pressures were measured only at the time of fetal delivery and were relative to air. The present in vivo preparation is probably a better reflection of the true changes. The increase in intratracheal pressure associated with tracheal obstruction is not surprising. As was mentioned earlier, the fetal lungs act as fluidsecreting organs up until near-term, and thereby contribute to the amniotic ~gid volume, zl-25 Active transport across the pulmQna~ epithelium eventually diminishes near term, in coordination with rising fetal cortisol levels, preparing the lungs for air-breathing. 26 Thus, it is expected that tracheal ligation or obstruction would result in fluid secretion in an enclosed space and would increase the intratracheal pressure. The pressure increase would continue until a plateau is reached 4 mm Hg in the present study. What is surprising is that such a small change in pressure is associated with a large difference in overall lung development. The gross changes associated with tracheal occlusion are comparable to those seen in other studies in terms of lung growth and control values. 5,8,9,2a,27,28 Lung weight and volume are increased by a factor of 2 to 3 with both tracheal obstruction and tracheal ligation. These lungs are obviously large for gestational age and appear normal histologically. As was shown in the present study, the effect of organ growth appears to be confined to the lung, because the heart and liver are not affected. This is consistent with what one sees in human congenital laryngeal atresia?
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Histological and morphometric analyses were used to compare lung architecture. From this it is evident that lung growth in response to tracheal obstruction results in an increased number of lung units--not simply an increase in the size of preexisting units. The airspace fraction was slightly higher in experimental lungs, and the alveolar wall was thinner, characteristics found in more mature lungs. 29 The alveolar surface area and alveolar number did not differ significantly between the two groups when expressed per unit of lung weight or volume. However, when expressed in terms of fetal body weight, the obstructed lungs show a two- to threefold increase in these values relative to those of controls. These changes are essentially proportional to those occurring at the macroscopic level. Most values obtained were consistent with those of other studies, 8,9 the exception being with alveolar number, probably a reflection of different methods in counting alveoli. The radial alveolar count (RAC) was found to be significantly different between obstructed and control lungs. This shows that alveolar complexity beyond the terminal bronchiole increases with this intervention.15.~6 To assess whether the tissue growth is related hyperplasia or hypertrophy, tissue DNA and protein content may be used. 8,9,24,3°,31 Increased mitosis will lead to an increase in tissue DNA levels. Increased production of interstitial matrix and cytoplasm will lead to elevated tissue protein levels. The ratio of tissue DNA to protein levels would be expected to remain constant if the balance between the number of ceils and the amount of extracellular matrix remains constant. In this study, both tissue DNA and protein levels between experimental and control lungs were unchanged when expressed per gram of lung tissue. The ratio of DNA to protein also was unchanged. However, because the experimental lungs were much larger, these values in terms of fetal body weight were significantly different, indicating that cell number and quantity of interstitium are elevated proportionally after tracheal obstruction. Analyses of the changes to the trachea show that damage occurs along the tracheal segment, between the tracheostomy site and the Swan-Ganz catheter balloon. The mucosa adjacent to the catheter tubing exhibits squamous metaplasia, probably from chronic rubbing by the catheter against the mucosa. At the balloon site the damage was moresevere, ranging from squamous metaplasia to frank necrosis and sloughing. It is comforting to note that even with the crude obturator used in this study, these changes were confined to the mucosa and submucosa, while
HASHIM ET AL
the cartilage was left relatively unaffected. Nevertheless it would be worthwhile to design a clinically applicable obturator that would minimize tracheal damage. It is evident from this study and others that prevention of fluid egress from the potential airspaces in the developing fetus results in accelerated lung growth.5,7-9,21 One proposed mechanism of action is the modulation of local growth factors via airspace pressure receptors. 8 An increase in lung fluid pressure may augment a local growth cascade. The effect on growth has been shown to confine itself strictly to areas of lung fluid retention, indicating the importance of fluid retention in this mechanism. 24 Systemically circulating growth factors would probably be ineffective without tracheal obstruction. Whereas pressure tends to cause necrosis of tissue (as is frequently seen with abcesses and tumor growth), tension can increase the mitotic activity in various tissues. Epithelial cell division in response to tension has been examined by several investigators. Increased mechanical stress applied to cultured fibroblasts was shown to increase the mitotic activity by speeding up the cell cycle. 32 Others observed the effect of applied tension in vivo on mouse skin and confirm increased mitotic activity in response to this intervention.33 Liu et al studied the effect of mechanical stretch on proliferation of fetal rat lung cells2 4 They concluded that mechanical forces act directly to stimulate fetal rat lung cell growth and that this effect is not mediated by prostaglandins or leukotrienes. The concept of tension affecting tissue growth has been applied to clinical practice for several years with the use of skin expanders. With fetal surgery becoming a reality in the clinical setting, methods of minimizing the surgical complications are being investigated. One of the gravest complications is preterm labor, which is often difficult to control medically. The uterine instability is believed to be secondary to the hysterotomy wound. 35 Thus, it seems logical that reducing the size of the wound may decrease this dreaded complication. Modern technology has allowed minimally invasive surgery with laparoscopic equipment, and this has recently been used in the experimental setting in fetal s u r g e r y . 3639 Our tracheal occlusion technique could easily be modified to incorporate the use of these techniques. ACKNOWLEDGMENT The authors are thankful to lain Vowels for his expert technical assistance in the animal care and experiments, and to Minh Duong for performing the DNA and protein assays.
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tions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658-665, 1984 22. Benson BJ, Kitterman JA, Clements JA, et al: Changes in phospholipid composition of lung surfactant during development in the fetal lamb. Biochim Biophys Acta 753:83-88, 1983 23. Fewell JE, Hislop AA, Kitterman JA, et al: Effect of tracheostomy on lung development in fetal lambs. J Appl Physiol 55:1103-1108, 1983 24. Moessinger AC, Harding R, Adamson TM, et al: Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86:1270-1277, 1990 25. Rethmeier HB, Egberts H: Transversal and longitudinal lecithin-sphingomyelin ratios in amniotic and lung fluids of fetal lambs. Am J Obstet Gynecol 122:593-596, 1975 26. Kitterman JA, Ballard PL, Clements JA, et al: Tracheal fluid in fetal lambs: Spontaneous decrease prior to birth. J Appl Physiol 47:985-989, 1979 27. Adzick NS, Outwate r KM, Harrison MR, et al: Correction of congenital diaphragmatic hernia in utero. IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985 28. Kitterman JA: Fetal lung development. J Dev Physiol 6:67-82, 1984 (review) 29. Docimo SG, Crone RK, Davies P, et al: Pulmonary development in the fetal lamb: Morphometric study of the alveolar phase. Anat Rec 229:495-498, 1991 30. Wigglesworth JS, Desai R: Use of DNA estimation for growth assessment in normal and hypoplastic fetal lungs. Arch Dis Child 56:601-605, 1981 31. Hosoda Y, Rossman JE, Glick PL: Pathophysiology of congenital diaphragmatic hernia. IV: Renal hyperplasia is associated with pulmonary hypoplasia. J Pediatr Surg 28:464-469, 1993 32. curtis AS, Seehar GM: The control of cell division by tension or diffusion. Nature 274:52-53, 1978 33. Squier CA: The stretching of mouse skin in vivo: Effect on epidermal proliferation and thickness. J Invest Dermatol 74:68-71, 1980 34. Liu M, Skinner SJ, Xu J, et al: Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol 263:376-383, 1992 35. Harrison MR, Golbus MS, Filly RA: The Unborn Patient: Prenatal Diagnosis and Treatment, chap 28. Philadelphia, PA, Saunders, 1991 36. Luks FI, Deprest J, Marcus M, et al: Carbon dioxide pneumoamnios causes acidosis in fetal lamb. Fetal Diagn Ther 9:105-109, 1994 37. Luks FI, Deprest JA, Vandenberghe K, et al: A model for fetal surgery through intrauterine endoscopy. J Pediatr Surg 29:1007-1009, 1994 38. Estes JM, Whitby D J, Lorenz HP, et al: Endoscopic creation and repair of fetal cleft lip. Plast Reconstr Surg 90:743-746, 1992 39. Estes JM, MacGillivray TE, Hedrick MH, et al: Fetoscopic surgery for the treatment of congenital anomalies. J Pediatr Surg 27:950-954, 1992