Journal of Surgical Research 148, 224–229 (2008)

The Effect of Tracheal Occlusion on Lung Branching in theRat Nitrofen CDH Model

Robert Baird, M.D.,* Nasir Khan, M.D.,* Helene Flageole, M.D.,* Mark Anselmo, M.D.,†Pramod Puligandla, M.D.,* and Jean-Martin Laberge, M.D.*,1

*Department of Pediatric Surgery, Montreal Children’s Hospital, Montreal, Quebec, Canada; and †Division of Respiratory Medicine,McGill University Health Center, Montreal, Quebec, Canada

Submitted for publication May 28, 2007

doi:10.1016/j.jss.2007.07.019

Background. Fetal tracheal occlusion (TO) hasbeen investigated as a treatment option for lunghypoplasia secondary to congenital diaphragmatichernia (CDH). TO increases lung size, but it is un-clear whether TO stimulates mature lung growth orsimply induces alveolarization without concomitantbronchial development. In this study, we character-ize bronchial branch development in fetal rats withCDH with or without TO through conventional histo-logical and morphometric analysis as well as lungcasting.

Materials and methods. Rat dams were gavaged ni-trofen at gestational day 9.5, and 3 to 4 fetuses per damunderwent fetal TO on gestational day 19 (term � 22days). Fetuses were sacrificed on day 21, the presenceof CDH was confirmed, and the lung weight to bodyweight ratio (LW/BW) was calculated. Lung casts of allresearch groups were created using liquid silicon andbronchial branches were quantified from lung periph-ery to carina.

Results. CDH fetuses had smaller LW/BW ratios anda lesser percentage (%) of airspace when compared tocontrols, and manifested less lung branching thancontrols. Fetuses treated by TO had a greater LW/BWratio and % airspace, but did not have a different num-ber of branch iterations. Fetuses with CDH and TOdemonstrated a restoration in LW/BW ratio to controllevels (P � 0.42), but the number of bronchial branchiterations remained less than control animals.

Conclusion. The results of this study suggest that TOin this animal model at gestational day 19 promotes dis-tal airway proliferation but does not reverse the under-

1 To whom correspondence and reprint requests should be ad-dressed at Department of Pediatric General Surgery, Montreal Chil-dren’s Hospital, 2300 Tupper Street, Room C-1137, Montreal, Quebec,

H3H 1P3, Canada. E-mail: jean-martin.laberge@muhc.mcgill.ca.

2240022-4804/08 $34.00© 2008 Elsevier Inc. All rights reserved.

development of bronchial branching seen in lung hyp-oplasia due to CDH. © 2008 Elsevier Inc. All rights reserved.

Key Words: fetal tracheal occlusion; congenital dia-phragmatic hernia; bronchial lung branching; nitrofen.

INTRODUCTION

Congenital diaphragmatic hernia (CDH) affects ap-proximately 1 in 2000 neonates, including stillbirths[1]. It has an incidence of 1 in 4000 to 5000 live births,and continues to be associated with significant morbid-ity and mortality [2]. Respiratory failure in infantswith CDH is primarily due to lung hypoplasia, a diag-nosis based on a constellation of histological findingsindicative of pulmonary immaturity. Comparison withnormal lungs confirms a marked reduction in the num-ber of bronchial divisions of hypoplastic lungs, which ismore severe on the side of the hernia [3] In addition,animals with experimental CDH display fewer alveoli,thickened alveolar walls, increased interstitial tissue,diminished alveolar air spaces, and a consequentialreduction in gas exchange surface area [4].

Tracheal occlusion (TO) has evolved as a potentialtreatment modality for prenatally diagnosed high-riskCDH. Although it has not proven superior to maximalpostnatal care in a randomized clinical trial [5], investi-gations of this technique have demonstrated a positiveeffect [6]. In animal models, the beneficial effects of TO onpulmonary hypoplasia have been demonstrated by im-provement in morphometric indices of the lung. Theseinclude a normalization of lung to body weight ratio (LW/BW) to non-CDH levels after TO, an increase in totalalveolar surface area, and a decease in alveolar septalthickness [7, 8]. Furthermore, parameters of lung func-

tion also demonstrate improvement after TO [9].

225BAIRD ET AL.: TRACHEAL OCCLUSION AND LUNG BRANCHING IN THE RAT CDH MODEL

The ability of TO to ultimately improve the outcomeof patients affected by severe CDH is predicated tosome degree on its capacity to promote mature lungbranching, an effect that has been poorly characterizedto date. While many of the histological changes seen inpulmonary hypoplasia are reversed after TO, it is cur-rently unclear if TO directly affects lung branching.The aim of our investigation is to assess the ability ofthe procedure to up-regulate pulmonary branching,both alone and in conjunction with a model of pulmo-nary hypoplasia.

MATERIALS AND METHODS

Animal Protocol

The animal protocol was approved by the McGill University ani-mal care committee (protocol no. 1224). Timed and dated pregnantrats (Charles River, Quebec, Canada) were identified by vaginalsemen plug, defined as day 0. These animals were transferred to theanimal care facility of the Montreal Children’s Hospital 5 d prior tothe start of experimental work to ensure optimal acclimatization.Animals were housed in groups of two per cage, and were allowedfood and water ad libitum. Dams were housed in standard laboratoryconditions with constant temperature (21° C) and 12-h day and nightcycles.

Study animals were gavaged nitrofen (2,4-dichlorophenyl p-nitrophenyl-ether-Chem Services, West Chester, PA) on fetal day9.5. The purity of the sample was determined to be greater than 98%with high performance liquid chromatography (Rohm and HaasCompany, Spring House, PA). Nitrofen (100 mg) was dissolved in 1 to2 mL of olive oil. A rigid metal tube was then inserted into theanimals’ esophagus to the approximate level of the upper stomachand the olive oil-nitrofen mixture was administered. There was noevidence of reflux or regurgitation after this procedure and all ani-mals tolerated it without complication. Control animals were fed bygavage with 1 to 2 mL of olive oil alone. All chemical handling wasdone in a fume hood with appropriate safety precautions.

TO

TO was performed on fetal day 19. Rat dams were administered axylazine/ketamine cocktail (1 cc/kg) until acceptable anesthesia wasachieved. Abdominal hair was removed with clippers and the surgi-cal site was prepared using a povidone-iodine solution. A midlinelaparotomy was performed, followed by exteriorization of the bicor-nuate uterus. The number of fetuses in each horn was highly vari-able, but generally ranged between four and eight fetuses. Thefetuses were carefully selected to undergo the occlusion. Factorsinfluencing which fetus underwent TO included intra-uterine posi-tioning and fetal orientation, the degree of uterine vascularity, andthe proximity of selected fetuses to avoid inadvertent injury to ad-jacent fetuses.

A 5-0 silk purse-string suture was placed in the overlying sac ofselected fetuses; one throw of the knot was placed. The uterus wasincised in a cruciform manner within the purse-string, identifyingthe outer uterine muscle, the underlying chorion and the innermostamnion. Penetration into the amniotic cavity was confirmed by ex-pulsion of amniotic fluid. The fetal head was then milked through theopening, followed by rapid cinching of the purse-string. Fetal expo-sure to the environment was minimized throughout the procedure tolimit fetal heat loss. In addition, warmed 0.9% saline was regularlyapplied to exposed fetal tissue to avoid desiccation.

A tethered suture was placed within the fetal mouth to hyperex-tend the neck and facilitate exposure. Using a floor-mounted surgical

microscope (Zeiss Inc., Oberkochen, Germany), a small vertical inci-

sion was made in the midline of the fetal neck and overlying tissuewas bluntly dissected to visualize the trachea. A surgical microclip(Pilling-Weck Inc., Markham, ON, Canada) was then placed on thetrachea under direct vision. Once accomplished, the uterine purse-string suture was loosened, followed by gentle upward traction onthe uterus as the fetal head was directed back into the uterus. Oneto two mL of warmed 0.9% saline were placed in the uterus tocompensate for lost amniotic fluid prior to tying of the purse-stringsuture. The uterus was returned to the abdominal cavity ensuring notorsion of the supplying vessels. Fetal viability was assessed byobserving its reaction to tactile stimuli prior to wound closure. Thelaparotomy incision was closed in two layers. Recovery was moni-tored through repeated assessments of maternal activity levels, oralintake and resumption of bowel habits. TO was performed in 3 to 4fetuses/dam. In addition, several non-nitrofen rat fetuses underwentsham operations. These animals underwent every step previouslydescribed except for application of the surgical clip on the exposedtrachea.

Sacrifice and Tissue Preparation

Fetal sacrifice was performed on day 21 of fetal life (term � 22days). Rat dams were kept alive until the end of the procedure tominimize insult to harvested fetuses. After opening the laparotomyincision, fetuses were removed from the uterus and viability wasassessed through the presence or absence of fetal movements.Fetuses were weighed and the abdomen was incised and the contentsremoved. The integrity of the diaphragm was assessed, and anyhernia was noted. The chest was then opened in the midline (caudalto cranial) until exposure of the proximal trachea was achieved. Atthis time, accurate placement of the tracheal clip was confirmed.

For bronchial casting purposes, lungs were left in situ and theproximal trachea was accessed using a 26-gauge needle throughwhich liquid silicon (RTV 3110; Dow-Corning Inc, Midland, MI) wasinjected. High pressure was used to inflate the lung airspaces withsilicon, until the material was uniformly distributed at the pleuralsurface of both lungs. These lungs were then dissected free andplaced in a 20% NaOH solution for 72 h. This process dissolved mostof the surrounding lung parenchyma, leaving a hardened siliconskeleton of the lung airspaces. The lung casts were then examinedunder a dissecting microscope (Zeiss Inc.).

For lung weight, histology and morphometry purposes, separatefetuses were used since this required dissection of the lungs beforeweighing and processing. Tissue preservation and fixation as well ashematoxylin and eosin staining was performed using standard tech-niques [10].

For both TO and tissue preparation, assignment of experimentalgroups was performed on a continuous basis and was dictated by thehigh mortality rate with TO.

Data Collection

Multiple images of individual lung casts were taken, and branchmaps were created using standard computer imaging software (Pho-toshop; Microsoft Inc., Redmond, WA) (Fig. 1). Ten points on theexterior surface of each left lung (known to be the most hypoplasticin left CDH) were chosen and all branch iterations were counted upto the carina. We used the Horsfield methodology of describing lungbranching from distal to proximal airways [11]. The Student’s t-testwas used to compare branch nodes measurements.

The percentage (%) airspace was calculated in four animals eachin three experimental groups as previously described [8, 12]. Twostandard hematoxylin and eosin histology images were examined peranimal by super-imposing a 16 � 24 grid on digital imagery (North-ern Eclipse; Empix Imaging, Inc., Mississauga, ON, Canada), yield-ing 384 intercepts. At each intercept, tissue or airspace was noted,resulting in a percent airspace for each slide. The mean interalveolardistance, or distance between airspace walls, was measured as the

mean linear intercept (Lm) in the same slides. This integer provides

with TO (not shown). (Color version of figure is available online.)

226 JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 2, AUGUST 2008

an index of alveolar size, and was calculated by dividing the totallength of five lines drawn across the lung section by the number ofintercepts encountered, as previously described [13, 14]. These re-sults were pooled within groups and compared using the unpairedStudent’s t-test (DATA Pro software; Microsoft). P � 0.05 was con-sidered significant in all cases.

RESULTS

Seven successive trials of rat dams were included inthis work, with a total of 234 fetuses involved in vari-ous experimental components. The efficacy of nitrofenin producing diaphragmatic hernia was 58.2% (64/110), the vast majority of which were left-sided. TOwas performed in 125 fetuses in total, with 52 survi-vors, for an overall success rate of 41.6%. An improve-ment in fetal viability was noted as experience with theprocedure increased. An overall difference in fetal sur-vival was noted in those animals that underwent TOalone (54.1%, 40/74) compared with animals that re-ceived both nitrofen and subsequent TO (23.5%, 12/51),P � 0.05.

Gross Measurements

Table 1 summarizes the data collected in controls,sham-operated animals and four experimental groups.Fetuses that had been exposed to nitrofen and were con-firmed to have developed a CDH (CDH�ve) had asmaller LW and a smaller LW/BW ratio than controls(P � 0.05 in both cases). Fetuses that had been exposed tonitrofen but did not develop a CDH (CDH–ve) had atrend toward smaller LW and smaller LW/BW ratiosthan controls. While fetuses that underwent TO did notmanifest a significant difference in lung weight, they diddemonstrate a greater LW/BW ratio than controls. Shamanimals had lung morphometric parameters that wereunchanged from controls, while CDH�ve fetuses thatunderwent subsequent TO demonstrated a restoration inLW/BW ratio to control levels (P � 0.42).

Lung Histology

Lung histology revealed qualitative differences be-tween control and experimental animals, with morealveolar airspaces in TO-affected animals as comparedto controls, and less airspace percentage in nitrofen-exposed fetuses. The measurements made on selectexperimental groups are summarized in Table 2. Thepercentage airspace was found to be significantly lowerin animals with confirmed CDH�ve as compared tocontrols (36.2% versus 49.2%, P � 0.05). Animals thatunderwent TO were found to have a greater percentairspace than controls (58.3% versus 49.2%, P � 0.05).These findings were corroborated by mean linear inter-cept (Lm) measurements. Lm was significantly lowerin CDH�ve animals (26.3 �m versus 30.3 �m, P �0.05), while TO animals demonstrated no difference inLm as compared to controls (33.1 �m versus 30.3 �m,

FIG. 1. Animal lung casts with branch-map overlay of left lung ongestational day 21. (A) TO (non-nitrofen exposed), which is similar tocontrol (not shown). (B) CDH positive, which is similar to CDH positive

P � 0.15).

227BAIRD ET AL.: TRACHEAL OCCLUSION AND LUNG BRANCHING IN THE RAT CDH MODEL

Lung Branching

Figure 1 demonstrates a digital image of a lungbranch cast with a branch-map overlay. Table 3 dem-onstrates the quantification of these lung casts. Con-trol animals were found to have a mean of 11.5 �/– 1.4lung branch nodes from the left lung periphery to thecarina (n � 8). Fetuses that were exposed to nitrofen inutero manifested a significant decrease in lung branch-ing. The CDH�ve group had 9.3 � 1.3 branch nodes(n � 5) and the CDH-ve group had 9.5 � 1.2 branchnodes (n � 4), P � 0.05 in both cases. There was nostatistical difference between these two groups. Ani-mals that underwent TO without exposure to nitrofendid not have a different number of branch iterations ascompared to controls (11.2 � 1.3, n � 4). Only twoanimals that were exposed to nitrofen and were subse-quently found to have developed a diaphragmatic her-nia underwent TO. These animals demonstrated lungbranching that was not statistically different than an-imals exposed to nitrofen alone (9.4 � 0.8, n � 2). Itshould be noted that these 2 fetuses are different fromthe 3 that were used to measure LW/BW (Table 1),since the latter requires removal of the lungs for weigh-ing and tissue processing, while the bronchial castswere performed in situ.

DISCUSSION

The development of branching patterns remains oneof the fundamental characteristics of organ develop-

TAB

Gross Measurements for

ControlN � 17

CDH�n � 15

CDHn �

LW (g) 0.14 � 0.02 0.09* � 0.024 0.11 �LW/BW (%) 3.79 � 0.35 3.07 � 0.52* 3.27 �

Note. All data presented as mean � SD.*Denotes P � 0.05 compared with controls.LW � Lung weight; LW/BW � Lung weight divided by body wei

hernia; CDH– � Nitrofen-exposed fetus that did not develop a diapSham TO in control animals (non-nitrofen exposed); CDH� and TOunderwent a TO.

TABLE 2

Measurements of Lung Histology and Morphometryfor Gestational Day 21 Fetuses

Controln � 4

CDH�ven � 4

TOn � 4

Airspace (%) 49.2 � 5.5 *36.2 � 6.1 *58.3 � 4.2Lm (�m) 30.7 � 2.2 *26.3 � 1.8 33.1 � 3.1

Note. All data presented as mean � SD.*Denotes P � 0.05 compared with controls.CDH� � Nitrofen-exposed fetuses that developed a diaphrag-

matic hernia; TO � Fetuses that have undergone TO alone; Lm �

Mean linear intercept.

ment and is highly conserved across species. Lungbranching allows the most efficient mechanism of gasexchange between the organism and the environment,a developmental process that occurs primarily in utero.This process has been shown to be restricted in neo-nates affected by CDH, although it remains unclearwhether techniques to improve in utero lung growthconcomitantly up-regulates lung branching. In hu-mans, proximal bronchial branching is complete by the16th week of development, with terminal bronchioleformation complete by the 28th week of gestation. Thedevelopment of alveolar sacs commences thereafter,and continues well into post-natal life. In rats, bron-chial branching during the canalicular stage of pulmo-nary development is generally complete by gestationalday 18 [15]. Alveolar development follows, with a peakin multiplication between 3 and 8 d of postnatal age.Expansion of the alveolar population continues until 1month of age [16, 17]. While investigators have exten-sively studied both the arterial branching and alveolarsize, number, and surface area of adult rat lungs [18, 19],to our knowledge ours is the first paper to quantify thebronchial branching of rat fetuses using lung cast tech-niques. The quantification of lung branching at earliergestational time-points would prove instructive, whiletechnically challenging and remains a potential targetof future investigation.

The process of lung branching seems at least par-tially dependant on the secretion of fluid from airwayepithelium in the developing lung. Occluding theegress of pulmonary secretions in whole organ cultureexplants increases the total number of airspaces, suchthat mechanical distending pressure resulting fromairway obstruction not only improves pulmonary archi-tecture but also accelerates lung development in vitro[20]. However, contradictory results have challengedthese initial observations. Heparin administration aswell as fluid restriction disrupts branching morphogene-sis in vitro [21, 22], while decreased intraluminal pres-sure significantly up-regulates lung branching [23].

Taken together, in vitro evidence appears to favor theconclusion that intraluminal fluid affects airway expan-

1

stational Day 21 Fetuses

e TOn � 8

ShamN � 5

CDH� and TOn � 3

.02 0.17 � 0.04 0.13 � 0.04 0.11 � 0.06

.59 5.44 � 0.82* 3.49 � 0.66 3.61 � 0.21

; CDH� � Nitrofen-exposed fetus that developed a diaphragmaticgmatic hernia; TO � Fetus that has undergone TO alone; Sham �

Nitrofen-fed animals that developed a diaphragmatic hernia and

LE

Ge

–v18

00

ghthra

�

sion and overall lung growth, but does not seem to direct

rwe

228 JOURNAL OF SURGICAL RESEARCH: VOL. 148, NO. 2, AUGUST 2008

lung branching per se. However, there remains a paucityof published data examining the in vivo effect of airwayocclusion on lung branching. Our investigation examinesbronchial branching in a rat model of pulmonary under-development as a consequence of teratogen-inducedCDH. TO shortly after the completion of bronchialbranching (day 19), was investigated in both normal andCDH animals.

CDH

The morphometric findings in animals exposed tonitrofen in our study are consistent with previous re-ports of morphometric changes in rat fetuses after ni-trofen administration [24–26]. Lung cast analysis re-veals a reduction in bronchial branch iterations inanimals treated with nitrofen, a sine qua non of pul-monary hypoplasia [3]. In addition, qualitative andquantitative histology comparing CDH�ve animals tocontrols demonstrates a reduction in airspaces, and adecrease in mean linear intercept consistent withgrowth retardation.

Others have demonstrated reduced branching mor-phogenesis after nitrofen administration in an organo-typic explant system. This supports the notion thatnitrofen interferes with early lung development beforeand independently from diaphragm development, sug-gesting that pulmonary hypoplasia in CDH may be theresult of a dual-hit from genetic/environmental factorsfollowed by a direct effect of the diaphragmatic hernia[27]. The comparison of animals exposed to nitrofen thatdeveloped a CDH (CDH�) with those that did not(CDH–) in our study indicates that it is the environment/genetic insult (in this case, nitrofen), that dominates thegross changes observed in this model of pulmonary hyp-oplasia. An independent effect of visceral herniation perse remains a possibility, however, and has been sug-gested by a difference in select mRNA expression be-tween CDH�ve and CDH–ve animals [28].

TO

The improvement in fetal survival after TO withsuccessive trials indicates that a significant learning

TAB

Lung Branching Data for

Controln � 8

CDH�n � 5

Lung branch (#) 11.5 � 1.4 9.3 � 1.3*

Note. All data presented as mean � SD.*Denotes P � 0.05 compared with controls.LW � Lung weight; LW/BW � Lung weight divided by body weigh

fetus that developed a diaphragmatic hernia; CDH– � Nitrofen-exposNitrofen-fed animals that developed a diaphragmatic hernia and unde

curve is associated with the performance of this proce-

dure. A plateau of 50% to 60% survival is reached afterseven trials of occlusion, an indication of the extrememorbidity of the procedure. Furthermore, the differ-ence in survival between those animals that under-went TO alone versus those that underwent nitrofenadministration followed by TO demonstrates the in-creased risk of fetal mortality after multiple insultsduring gestation.

While TO increases lung weight and the LW/BWratio as observed by other groups [26, 29], it does notup-regulate bronchial branching in control animals norreverse the underdevelopment of bronchial branchingseen in nitrofen-induced CDH animals. This is likely dueto the temporal relationship between nitrofen adminis-tration and TO. The earlier nitrofen-induced insult re-duces the branch formation during the pseudoglandu-lar and canalicular stages of lung development (day9.5), whereas TO is performed during the canalicular-saccular stage (day 19) and only influences the distalproliferation of alveolar units. The timing of TO is alsoan important factor in its effect on pulmonary devel-opment. TO in rabbits during the late pseudoglandularstage results in an initial 3-d stagnation of growth andsubsequently a dramatic acceleration of growth,whereas TO during the early terminal sac stage inducesan immediate and sustained moderate increase in lunggrowth [30]. The observation that TO increases the per-cent airspace without altering the Lm suggests that thenumber of alveolar units increases as a consequence ofthe intervention. Alveolar distension does not appear tosignificantly contribute to the increase in observedairspace—consistent with previous observations [7, 31].

We had hypothesized that TO may have the ability to“de-differentiate” the developing lung such that bron-chial branching may have occurred beyond the gesta-tional age at which it should be complete. This does notappear to be the case in this experimental model. Therewere only two animals in the CDH � TO group, butboth had a similar number of branches to the CDHgroups. Adding the fact that the four TO animals had anumber of branches similar to controls, it is unlikelythat sacrificing more animals would have changed the

3

stational Day 21 Fetuses

CDH–ven � 4

TOn � 4

CDH� and TON � 2

9.5 � 1.2* 11.2 � 1.3 9.4 � 0.8*

O � Fetus that has undergone TO alone; CDH� � Nitrofen-exposedfetus that did not develop a diaphragmatic hernia; CDH� and TO �nt a TO.

LE

Ge

t; Ted

results.

229BAIRD ET AL.: TRACHEAL OCCLUSION AND LUNG BRANCHING IN THE RAT CDH MODEL

While we may conclude from our lung branchingresults that TO at gestational day 19 does not up-regulate gross lung branching, it remains conceivablethat occlusion at an earlier stage in development mayresult in improved bronchial branch development.While the process of bronchial branching is nearly com-pleted by day 19 in the fetal rat, it corresponds reason-ably well to the 22nd to the 28th week of human ges-tation that has been used for TO in human trials [5, 6].Attempting TO in fetal rats earlier than gestationalday 19 would not be relevant for human applicationand therefore the increased morbidity and technicalchallenge do not appear warranted. The observed lackof effect on bronchial development agrees with recentclinical evidence that has demonstrated no improve-ment in survival in fetuses treated by TO with evi-dence of extremely underdeveloped lungs as mani-fested by an in utero lung-to-head ratio � 0.8 [6]. Ourexperimental demonstration of no effect may thereforehelp to explain the clinical observation that extremelyhypoplastic lungs lack sufficient bronchial develop-ment for TO to prove effective, despite the subsequentalveolar proliferation.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Georgia Kalavritinos and theMontreal Children’s Hospital Research Institute for their support.

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