fetal lung growth after tracheal ligation is not solely a pressure phenomenon

Fetal Lung Growth After Tracheal Ligation Is Not Solely a Pressure Phenomenon

By Konstantinos Papadakis, Franqois I. Luks, Monique E. De Paepe, George J. Piasecki, and Conrad W. Wesselhoeft, Jr

Providence, Rhode Island

l Fetal tracheal ligation increases lung growth in utero, making it potentially applicable for antenatal treatment of diaphragmatic hernia. This phenomenon has been ascribed to increased intratracheal pressure, which activates as yet unidentified pulmonary stretch receptors. The purpose of this study was to determine whether the composition of lung fluid has any effect on fetal lung development after tracheal obstruction. Six sets of fetal lamb twins underwent tracheal ligation with placement of intratracheal catheters at 122 days’ gestation (term, 145 days). In group 1 (n = 6). tracheal fluid was aspirated daily, measured, and replaced with equal volumes of saline. Their respective twins (group 2, n = 6) had daily reinfusion of their own tracheal aspirates. lntratracheal pressure was recorded daily in both groups. Unobstructed fetal lambs (n = 7) were used as negative controls. Animals were killed on postoperative day 14 (136 days). Lungs were weighed, perfusion fixed at 25 cm H20, and processed for standard morphometric analysis. Intratra- cheal pressure remained between 3 and 5 torr in both experimental groups throughout the entire postoperative period. In all 12 experimental fetuses, tracheal ligation resulted in an almost threefold increase in lung fluid volume by day 1; a slight decrease at a mean of 2.4 days; and a second surge from day 4 on. Lung fluid volume was significantly higher in group 2 than in group 1 at all measured time points (P < .05, Wilcoxon rank sum test) except on days 3,4, and 8 (P= .06). Lung weight per body weight (LW/BW) at delivery was 0.045 2 0.008 in group 1, not significantly different from unobstructed controls (0.038 ? 0.006). LW/BW in group 2 was 0.055 2 0.010, significantly larger than either group 1 or control (P < .05, single factor analysis of variance). Air space fraction was comparable between the three groups. Alveolar numerical density was significantly lower in groups 1 and 2 than in unobstructed controls (P < 0.05). Replacement of tracheal fluid with saline inhibits the lung hypertrophy seen after tracheal ligation. This phenomenon therefore appears more dependent on tracheal fluid growth factors than on increased intratracheal pressure after obstruction. The immediate decrease in net lung fluid production after saline exchange suggests that these humoral factors play an important role in the initiation of lung cell proliferation. Copyright o 1997 by W.B. Saunders Company

INDEX WORDS: Fetal surgery, fetal lung growth, congenital diaphragmatic hernia, tracheal obstruction.

I NFANTS BORN WITH congenital diaphragmatic hernia (CDH) often die of causes secondary to

pulmonary hypoplasia. This hypoplasia is, in part, a consequence of compression by the herniated viscera during gestation, and the severity of the hypoplasia appears to be a function of the degree and duration of compression.‘-“ Recent experience has shown that in

Journal ofPedIatric Surgery, Vol 32, No 2 (February), 1997: pp 347-351

utero tracheal obstruction can counteract the pulmonary hypoplasia associated with CDH,5.6 making it a potential modality to treat CDH antenatally.5,6 This approach may lend itself particularly well to minimally invasive fetal surgery,7-9 or even to fetal tracheoscopy.‘O.”

Lung growth after tracheal obstruction, once believed to result in overdistended, hypertrophic lungs,‘?.i3 seems to represent true lung hyperplasia5 Very little is known about the exact mechanisms of lung growth after tracheal obstruction. A pressure phenomenon has been postulated, through as yet undefined pulmonary stretch receptors.6,14 There is at least some in vitro evidence that humoral factors present in lung fluid play a role in this exaggerated lung growth.‘” To test the importance of tracheal fluid composition, we created an in vivo model of tracheal obstruction whereby fetal lung fluid was replaced by saline.

MATERIALS AND METHODS

Ttme-dated pregnant ewes t 122 days, term, 135 days) wtth twm gestations underwent mtdlme laparotomy under general halothane anesthesta (0.52.0% in 100% 01) after intravenous mductton wtth ketamine ( 1 g) A small hysterotomy was made transversely near the uterme horn with exteriortzatton of the fetal head and neck. The uterine wall was secured around the fetal neck wtth Alhs clamps to mmtmize amniotic flutd loss. The fetal trachea was then exposed and atraumatt- tally clamped to avotd egress of tracheal fltnd. A tracheotomy was made. and a 19.gauge inner dtameter CID) flanged tygon catheter was Inserted mto the dtstal trachea and secured wtth 4-O polypropylene (Prolene. Ethicon, Johnson & Johnson, Somerville, NJ). Trachea1 ltgatton was performed wtth #2 sdh cephalad to the tracheotomy. A 17.gauge ID ammottc catheter. fitted wtth a cage. was attached to the neck at the level of the trachea1 catheter. The fetal head and neck were returned to the uterus. Antibiotics (ampicillm. 700 mg and chloramphemcol. 250 mg) were added to the amniottc fluid. The hysterotomy was closed using a two-layer continuous closure and the catheters were “Wttzeled.” The abdominal wall was closed after ensurmg adequate slack on the catheters. The catheters were extertortzed mto a pouch on the rtght flank of the ewe.

From thr D~~wott of Prdrarrrc~ Surgey and the Department of

Pathology, Bro+r II Unrver.~~ School of Medicme. Husbro Children k

und Rhode Island Hosprtals. Provrdmce. RI

Presented ut the 27th Ann~ui Mretmg of the Amertcun Pediatrrc

Sur,qtal Assocrcrtron. Sun Diego, Cul$wniu. Mu 20-23. 1996.

Supported m put b? Grunt No. 5177 from the Rhode Island

Foundatro,~.

Address reprrnt reyur3t.t to Frutywr 1. Luhs. MD, DllYsion of

Pedintrrc Surgery, Hubro Children i Hospital, 2 Dudley St, Surte 180.

Provrdence. RI 02905.

Cop>riyht 8~ 1997 by WE Surtnders C’ompun~

0022-3468/97/3202-0040$03 00/O

347

348 PAPADAKIS ET AL

Twelve fetal twins were divided mto two groups. One fetus of each pregnant ewe (group 1, n = 6) underwent intraoperative, followed by

daily aspiration of lung thud, which was exchanged milliliter for millihter with room temperature salme. Their respecttve twins (group 2, n = 6) underwent dally aspiration and reinfusion of their own lung fluid. The amount of the daily fluid asptrates was recorded. Both groups had daily measurements of mtratracheal and ammotic pressures (Grass PI-l pressure transducer and Dash IV, 4-channel preamplifier-recorder, Astro-Med. West Warwick, RI) True intratracheal pressure was defined as amniotic minus tracheal pressure.th After the daily manipulatton, 250 mg of ampictllin and 250 mg of chloramphemcol were Infused into the amniotic catheter. Seven nonoperated fetal lambs were used as controls.

At 136 days’ gestabon (postoperattve day 14) the ewe underwent Cesarean section under the same anesthetic conditions. The previous hysterotomy was used to deliver the fetuses. Fetal breathmg was prevented by placing a surgical glove over the fetus’ head. The fetuses were killed by injection of a euthanasia solution (3 mL of Beuthana- sia-D Special, Shermg-Plough Ammal Health Corp., Kenilworth, NJ) into an umbilical vein after clamping of the cord. The fetus’ body weights were recorded, and the fetal lungs and trachea were removed en bloc.

Histology and Morphometric Analysis

The wet weights of the lungs were determmed. Fixatton was accomplished by tracheal instillation of 10% buffered formaldehyde mamtained at a pressure of 25 cm Hz0 with the lungs immersed in a beaker containing buffered fixative The lungs of respective twins were fixed in parallel to eliminate fixation variabihty. After at least 7 days’ fixation at 2 1 “C the trachea was clamped and the volume of the lungs was estimated by the volume displacement method. The lungs were cut m standardized sections (3 to 5 mm thick) and random blocks from each lobe were embedded in parafbn. Hematoxylin and eosin (H&E)-stained slides (4 m thick) were prepared from each paraffin block (four per animal).

Lung tissue sections were analyzed morphometrtcally using a computerized image analysis system: microscope interfaced vta a charge-coupled device (CCD) video camera (KP-161, Hitachi USA, Woodbury, NY) to a Power Macintosh 7 100/8OAV (Apple Computer, Cupertino, CA) eqmpped with software for image analysis (Image 1.59 for Macintosh. National Institutes of Health. Bethesda, MD). The system was programmed to measure lung atrspace fraction. The number of alveoli per unit area was estimated by counting alveolar profiles within the test area, whereby an alveolus was defined as an airspace either entirely or partially enclosed by respiratory epithelmm. Forty fields were counted for each lung at a magnification of 200X ( 10 fields for each of the right apical, right diaphragmatic, left apical. and left diaphragmatic lobes). Data were corrected for fetal body weight where appropriate to allow for comparison between groups.

Statistical analysts was performed using the Wilcoxon rank sum test (paired compartson of groups 1 i* 2). or single-factor analysis of variance (ANOVA) for compartson of three nonparametric groups. Where appropriate, values are expressed as mean + standard deviation. A P value of less than .05 was consrdered statistically significant. All procedures and protocols were approved by the Brown Umverstty Animal Care and Use Committee.

RESULTS

Intratracheal Pressure and Lung Fluid Volume

Intratracheal pressure (when corrected for amniotic pressure) increased from 1.9 -C 1 .O torr before ligation to 3.7 -+ 0.8 torr by the second postoperative day. Thereaf- ter, intratracheal pressures remained stable, ranging from 3 to 5 torr throughout the study period. There was no

statistically significant difference in pressure between the two groups. When lung fluid production was corrected for body weight at birth, tracheal ligation resulted in an almost threefold increase in lung fluid volume by day 1 (from 11.7 ? 4.8 to 28.4 ? 9.3 mL/kg in group 1 and from 12.1 ? 4.1 to 36.8 + 8.5 mL/kg in group 2); a slight decrease at a mean of 2.4 days (nadir 24.6 ? 4.8 in group 1,3 1.4 _f 7.3 mL/kg in group 2); and a second surge from days 4 to 10. Lung fluid volume was significantly higher in group 2 than in group 1 at seven of 10 measured time points (P < .05, Wilcoxon rank sum test; Fig 1). Catheter malfunction in several animals toward the end of the experimental period precludes meaningful data interpreta- tion beyond day 10 postligation.

Lung Weight

There was no statistical difference in lung weight to body weight ratio (LW/BW) between group 1 (ligated/ saline) and nonligated controls (P > .13, single factor ANOVA). LW/BW was significantly higher in group 2 (ligated/unaltered lung fluid) than in either group 1 (P < .05) or control (P < .005). Results are summarized in Table 1.

Histology and Morphometry

Histologically, the lungs of age-matched control animals showed well-developed alveolar sacs and ducts, lined by thin alveolar septa (Fig 2A). In both experimental groups, the air spaces appeared dilated and the alveolar septa attenuated (Figs 2B and C). Air space fraction did not differ significantly between the three groups (Table 1). Alveolar numerical density (number of alveoli per cubic centimeter of lung tissue) was significantly lower in both ligation groups than in controls (P < .05, single factor ANOVA).

Fig 1. Average net lung fluid volume after tracheal ligation. Group 1, ligated/saline; group 2,ligated/unaltered fluid. l P c .05, Wilcoxon rank sum test.

ROLE OF LUNG FLUID IN FETAL TRACHEAL LIGATION 349

Table 1. Lung Weight and Morphometric Data

Control Group 1 Group 2 PValue

Lung weight/body weight 0.038 5 0.006 0.045 t 0.008 0.055 + 0.010* <.05 Air fraction (%) space 74 8 + 6.6 74.4 t 5.1 75.4 + 2.5 Not significant Alveolar numerical density (r106/cm3) 82.45 5 26.14t 57.01 2 11.46 44.74 -c 11.22 c.05

NOTE. Group 1, ligated/saline; group 2, ligated/unaltered fluid; control, unobstructed trachea

*Group 2 vgroup 1 and control.

tGroup 1 and group 2 vcontrol, single-factor ANOVA.

DISCUSSION

The exact mechanism by which tracheal ligation promotes lung growth in the developing fetus is not known. The present study evaluates the importance of tracheal fluid composition in this process. Here. tracheal ligation was performed in fetal twins under identical pressure conditions. When tracheal fluid was preserved, tracheal obstruction resulted in significantly increased lung weight, as expected from previous reports.5%6.“.‘6 Replacement of tracheal fluid with saline resulted in significant inhibition of this lung growth phenomenon. Indeed, lung weight after ligation and saline replacement was not significantly different from that of unobstructed controls. This indicates that tracheal fluid composition, rather than intratracheal pressure, is critical in promoting pulmonary growth. Air space fraction was similar in all three groups, confirming that the rise in lung weight and lung volume after tracheal ligation is caused by tissue growth, not just overdistension by an elevated intratracheal pressure.6

DiFiore and Wilson’s have previously shown that lung fluid after tracheal ligation is mitogenic to pneumocytes in vitro. Although the actual cell proliferation and increase in lung fluid production is indeed likely to be mediated by one or more growth factors,‘7.‘n their study did not establish whether humoral factors are primary or secondary effecters. Elevated intratracheal pressure and pulmonary stretch could still have initiated this increase in growth factor concentration. In the present experiment, blunting of the lung fluid volume curve in group 1 (ligation/saline) was noted as early as day 1. despite intracheal pressures similar to those in group 2 (ligation/ unaltered tracheal fluid). Thus. fluid composition seems to play a role not only in subsequent lung growth, but also in the initial signal to cell proliferation after tracheal ligation.

The increased intratracheal pressure may still have an effect on the ultimate lung architecture. When compared with control lungs. the alveolar septa in both experimental groups were shortened, causing coalescence of the alveolar spaces. These alveoli are poorly defined and appear larger. This would explain the observed reduction in alveolar numerical density (number of alveoli per mm3 of lung tissue) after tracheal ligation, a finding that seems at odds with the concept of functional lung growth. The

Fig 2. Photomicrographs of random fields of respiratory tissue from (A) control animals, (El group 1 (2-week tracheal ligation, daily exchange of tracheal fluid with saline), and IC) group 2 (2-week tracheal ligation, no exchange of tracheal fluid). (A) In control lungs, the alveoli are well demarcated by prominent septations. (B,Cl In ligated lungs, the septa appear shortened and the alveoli less well defined. (H&E, original magnification ~400).

350

very definition of alveoli has been subject to contro- versy. I9 and has rendered reproducibility of morphometric results difficult.6.“.20

The lung fluid volume curve after tracheal ligation may offer insight into the time course of lung growth after tracheal obstruction. Within 24 hours after tracheal ligation, net lung fluid production increases threefold, from 10 to 15 mL/kg to 30 to 45 mL/kg. If fluid production were to continue at the predicted preligation rate of 3.4 to 4.5 mL/kg/h21-‘3 however, tracheal fluid volume would have risen from 10 to 15 mL/kg to 90 to 120 mL/kg within the first day-a six- to tenfold increase. Thus, net lung fluid production actually de- creases after tracheal ligation; an increase is not seen until 3 days later. This initial reduction in net fluid production could be the result of reduced secretion or accelerated reabsorption of lung fluid; both could partially be caused by a catecholamine or cortisol surge after surgical stress, similar to what is observed intrapartum.‘4,25 This effect is unlikely to continue beyond the immediate postoperative period, however. Rather, the sustained fall in net fluid production during the first 72 hours after tracheal ligation appears to be the result of the obstruction itself. The noted elevation in intratracheal pressure may partially be respon- sible for this, because high intratracheal pressure is known to impair fluid secretion and promote reabsorption.16

Why, then, does net fluid production rise after 3 days, if

PAPADAKIS ET AL

hydrostatic pressures within the tracheobronchial tree remain elevated throughout the entire 2-week period? This could be a reflection of increased secretory cell mass within the lung, suggesting that cell proliferation becomes apparent 2 to 3 days after tracheal obstruction. Indeed. Hooper et a127 reported that two-thirds of the increase in pulmonary DNA content occurred between 2 and 7 days postligation. Others have also commented on the short time required for lung growth to occur after tracheal obstructionh~“,**

The principle of lung growth after tracheal obstruction is now well recognized, and preliminary reports in humans have suggested its potential usefulness in the antenatal treatment of CDH.zx.29 It is important that the mechanisms involved in this process be better under- stood, however, before clinical application becomes widespread. Knowledge of the physiological and molecu- lar basis of normal and accelerated pulmonary growth may enable us, in the future, to upregulate, or turn off, pulmonary growth more selectively after tracheal obstruction. It may become possible to avoid tracheal obstruction altogether. by acting directly on the regulatory mechanisms themselves.

ACKNOWLEDGMENT

The authors express their gratttude to Mohammed Traore for hts technical assistance.

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fetal lung growth after tracheal ligation is not solely a pressure phenomenon

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