Lung development following diaphragmatic hernia in the fetal rabbit

J. Wu1, H. Yamamoto1,5, E. Gratacos1,3, X. Ge1, E. Verbeken2, K. Sueishi6, S. Hashimoto6, K. Vanamo1,7, T. Lerut1,4 and J. Deprest1,3,8

1 Center for Surgical Technologies, Faculty of Medicine, Katholieke Universiteit Leuven, 2 Departments of Pathology, 3 Obstetrics & Gynaecology and 4 Thoracic Surgery, University Hospital `Gasthuisberg', Leuven, Belgium, 5 Department of Pediatric Surgery, Kumamoto University School of Medicine, Kumamoto, 6 Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan and 7 Department of Pediatric Surgery, Kuopio University Hospital, Kuopio, Finland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diaphragmatic hernia was created in 39 rabbit fetuses on day 23 of gestation. Fifteen fetuses underwent a sham thoracotomy (SHAM). Thirty-nine non-operated littermates served as internal controls (CTR). Fetuses were harvested by Caesarean section on days 25, 27, 29 and 30 of gestation. Pulmonary response was evaluated by lung to body weight ratio (LBWR), morphometry, and density of type II pneumocytes. No difference was found between CTR and SHAM fetuses at term. CDH fetuses had smaller lungs (LBWR 0.014 ± 0.004 versus 0.030 ± 0.04 in CTR, P < 0.0001), a less complex acinus [mean terminal bronchial density (MTBD) 1.786 ± 0.408 versus 0.917 ± 0.188, P < 0.0001], thicker alveolar septa [mean wall transection length (LMW) 0.0221 ± 0.008 versus 0.0142 ± 0.002, P = 0.0003], and a lower type II cell count (144.5 ± 19.33 versus 216.2 ± 27.85 per high power field, P < 0.0001). The differences in MTBD and LMW were significant from gestational day 25 onwards, and the differences in type II cell count from day 27 onwards. Surgical diaphragmatic hernia in rabbit fetuses in the late pseudoglandular phase reproduces many features of the pulmonary hypoplasia associated with human congenital diaphragmatic hernia, including the delayed maturation. The effects are present within 2 days following experimental diaphragmatic hernia and progress over time.

Key words: animal models/congenital diaphragmatic hernia/fetal surgery/lung development/pulmonary hypoplasia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Congenital diaphragmatic hernia (CDH) affects 1 in 3000–5000 human births and is associated with a high mortality, mainly due to pulmonary hypoplasia and persistent pulmonary hypertension (Areechon and Reid, 1963Go; Harrison et al., 1994Go). In-utero intervention has been proposed to correct the pulmonary hypoplasia prenatally. Animal models have demonstrated the feasibility of surgical repair, and, more recently, tracheal occlusion (TO) (Alcorn et al., 1977Go; Hedrick et al., 1994Go; Flageole et al., 1998Go). Anatomical repair has been abandoned in humans because of poor results (Harrison et al., 1993Go). Early experience with TO is promising (Harrison et al., 1998Go; Albanese et al., 2000Go), although the optimal timing of the procedure and the characteristics of the resulting lung are still under intensive experimental research.

Different animal models have been used in the study of CDH and its potential in-utero therapy, including sheep, rabbits and rats (Wilcox et al., 1996Go; Fauza et al., 1994Go; Kitano et al., 1998Go). In the rat the pathology is induced by means of nitrofen administration, while fetal lambs are commonly used as models for fetal surgery. Although all mammalian species undergo the same stages of lung development, the duration and timing of these stages vary greatly in relation to gestational age. In the rabbit, the pseudoglandular phase extends to day 23–24 of gestation, covering ~75% of gestation. The three remaining phases are rushed through in 3–4 days each (Kikkawa et al., 1968Go; Pringle, 1986Go). Alveolization starts prior to birth, to be completed postnatally. In this respect, rabbits resemble humans more closely than the other animal models frequently used to study CDH. In sheep, most alveoli are present at birth, and in rats alveolization begins on the fourth postnatal day (IJsselstijn and Tibboel, 1998Go). Rabbits are also relatively inexpensive, non-seasonal in their mating habits, have a short gestation period and a large litter size, which makes them suitable for studies requiring surgical manipulation of the fetus.

Most experimental studies have focused on pulmonary outcome at term. In other words, few studies have attempted to assess the evolution of pulmonary hypoplasia after experimental diaphragmatic hernia (CDH). This background knowledge, however, is essential to interpret the results of prenatal interventions performed at different gestational ages. The aim of this study was to evaluate the effect of surgically induced diaphragmatic hernia on lung growth and maturation in the fetal rabbit, prior to embarking on further studies on the effect of TO on hypoplastic lungs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and study groups
Thirty-nine time-mated pregnant New Zealand rabbits were operated on day 23 of gestation (term = day 32). In 15 of these animals, diaphragmatic hernia was created in one ovarian end fetus (CDH), while the fetus at the other ovarian end underwent a left thoracotomy without CDH (SHAM), in order to rule out effects of fetal surgery per se. The fetuses were harvested by Caesarean section on day 30 of gestation in order to ensure no does had delivered: as morphometry was the most important outcome measure, animals had to be killed prior to birth, which could only be guaranteed 2 days prior to term. In each doe, a non-operated littermate of equal size to the operated ones served as a control (CTR). In the remaining 24 animals, CDH was created in only one ovarian end fetus on day 23 and harvesting took place by Caesarean section on days 25 (n = 8), 27 (n = 8), and 29 (n = 8) of gestation (CDH). In each doe, two non-operated littermates of equal size to the operated one served as controls (CTR). Animals were treated according to current guidelines on animal wellbeing, and the Ethical Committee for Animal Experimentation of the Faculty of Medicine of the Katholieke Universiteit Leuven approved the experiments.

Anaesthesia and surgical procedure
Prior to surgery, the animals were housed in separate cages at normal room temperature and normal daylight, with free access to food and water. On the day of operation, they were premedicated with ketamin 50 mg/kg i.m. (Ketalin®; Apharmo, Arnhem, The Netherlands), promazinum hydrochloridium 5 mg/kg i.m. (Prazine®; Libamedi, Brussels, Belgium), and penicillin G 300 000 IU i.m. Anaesthesia was maintained with 2-5% halothane in oxygen 1 l/min. Maternal heart rate and oxygen saturation were monitored with a pulse oximeter (Nellcor® N-20P; Nellcor Inc., Haasrode, Belgium).

The animals were placed in the supine position and the abdomen was shaved under continuous vacuum aspiration, disinfected with povidone iodine (Iso-Betadine®; Asta Medica, Brussels, Belgium) and draped in a sterile fashion. The pregnant uterus was exposed through a lower midline laparotomy.

Uterine interventions were performed with micro-instruments under an operating microscope (Carl Zeiss, Oberkochen, Germany; magnification x5–25). After determining the fetal position by gentle palpation, a 1 cm longitudinal incision was made on the antimesometrial side of the uterus. The membranes were fixed to the uterine wall with four 6–0 sutures (Prolene®; Ethicon, Dilbeek, Belgium). The left side of the fetal chest was exposed by gentle manipulation and fixed to the uterine wall with a single 6–0 suture. The diaphragm was exposed through a low left lateral thoracotomy using purpose-designed retractors and the membraneous part was opened with scissors. The thoracotomy was closed in one layer with 6–0 interrupted sutures. In sham operations, the diaphragm was exposed through an identical thoracotomy, but not opened.

After removing the stay suture, the fetus was gently manipulated back into the uterine cavity and the hysterotomy was closed with a running 6–0 Prolene suture. The uterus was replaced into the abdominal cavity and the abdomen was closed in layers with 3–0 polyglactine (Vicryl®; Ethicon) for the fascia and subcutaneous tissue and intracutaneous 2–0 nylon (Ethilon®; Ethicon) for the skin. Medroxyprogesterone acetate 4.5 mg i.m. (Depo-Provera®; Pharmacia-Upjohn, Puurs, Belgium) was given postoperatively for tocolysis. Preoperative daily care was resumed after the operation.

Second-look operation and tissue sampling
On the appropriate day of gestation the does were killed with an i.v. bolus of a mixture of embutramide 200 mg, mebezonium 50 mg and tetracain hydrochloride 5 mg (T61®; Hoechst, Brussels, Belgium). Fetuses were delivered by Caesarean section. Macerated and stillborn fetuses were recorded as non-survivors and excluded from further analysis. The total body weight, the wet lung weight, the liver weight, and the kidney weight of each fetus were measured. The lung weight to body weight ratio (LBWR) was calculated. The lung volume (LV) was assessed by the volume displacement method. The trachea was cannulated with a 22 gauge i.v. catheter, and the lungs were immersed in and inflated with 10% neutral buffered formalin solution at 25 cm H2O pressure for 24 h. The right and left lungs were then separated, weighed, and embedded in paraffin.

Lung morphometry and type II pneumocyte density
The paraffin-embedded lungs were cut into 5 µm sections, and stained with haematoxylin and eosin for morphometry. Two observers (X.G. and E.V.), blinded to the experimental surgery, evaluated the morphometric parameters in 40 randomly selected non-overlapping lung fields for each fetus. The parameters included mean terminal bronchial density (MTBD), linear intercept (LM), septal thickness (LMW) and type II pneumocyte density per unit area. MTBD is a morphometric method based on the principle that the number of terminal bronchioles in a given high power field is inversely proportional to the number of alveoli supplied by each bronchiole (Verbeken et al., 1994Go). LM is an index of alveolar size (Verbeken et al., 1992Go). LMW is an index of the thickness of the septa of the parenchymal air-spaces (Dolhnikoff et al., 1995Go). MTBD was measured at a magnification of x100, and LM and LMW at x200.

To obtain type II pneumocyte density, the tissue sections were processed for avidin-biotin complex immunoperoxidase staining. A polyclonal anti-rabbit surfactant protein (SP)-A antiserum reacting with type II pneumocytes and Clara cells (typically found in lungs) was used (Asabe et al., 1994Go). Sections were treated with 3,37-diaminobenzidine tetrachloride (Sigma Chemical Co., St Louis, MO, USA), slightly counterstained with haematoxylin, cleared, and mounted. Specificity was confirmed by exclusion of the primary antibody in the preparation. The density of SP-A positive cells was determined by counting positively stained cells per field in 40 random non-overlapping fields (magnification x20). The result was converted to the number of surfactant protein-A positive pneumocytes per mm2.

Statistical methods
Statistical analysis was performed with one-way analysis of variance (ANOVA) for multiple comparisons (Tukey's test) or Student's t-test. Values are expressed as mean (SD) where appropriate. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of surgery and gross anatomical findings
The body weights, the LBWR, and the survival of fetuses in each study group are displayed in Table IGo. Figure 1Go shows the change in LBWR with day of gestation at death. A diaphragmatic hernia was present in all surviving fetuses in the CDH group. The mean total body weights were similar in all groups on each day of gestation. CDH fetuses had a significantly lower LWBR compared with control fetuses from gestational day 25 onwards, and this difference increased with gestational age (Table IGo, Figure 1Go). In contrast, the LBWR in the SHAM group was similar to controls on day 30 of gestation.


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Table I. Survival, body weight, and lung weight body weight ratio (LBWR) in fetal rabbits after in-utero surgical diaphragmatic hernia
 


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Figure 1. Lung to body weight ratio (LBWR). Values represent mean ± SD of control (CTR) and diaphragmatic hernia (CDH) fetuses. *P < 0.01 versus controls.

 
Lung morphometry and density of type II pneumocytes
The results of morphometry and type II pneumocyte densitometry are presented in Table IIGo. The lungs of fetuses in the SHAM and CTR groups were morphometrically identical on day 30 of gestation (data not shown). The lungs of fetuses with CDH were, however, significantly different from the CTR lungs on all gestational days assessed. In CTR animals MTBD, reflecting the number of terminal bronchioles, declined near term. In CDH fetuses, the MTBD was significantly higher than in controls on any given gestational day and lacked a terminal decline. LMW, an index of the septal thickness of the parenchymal airspaces, declined towards term in both CTR and CDH fetuses, but was significantly higher in CDH fetuses on any given day of gestation. LM, a measure of parenchymal airspace size, declined towards term in both control and CDH fetuses, with no difference between the groups (Figure 2Go). Very few type II pneumocytes were present on day 25 of gestation in both CTR and CDH lungs. From 27 days onwards, however, the density of type II pneumocytes increased slower in CDH lungs, with a progressively increasing difference as compared to controls (Figure 3Go). The density of type II pneumocytes in the SHAM group was similar to that of controls (data not displayed).


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Table II. Results of morphometry of the left lung in rabbit fetuses with diaphragmatic hernia versus normal controls
 


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Figure 2. Light microscopic appearance of fetal lungs at 24 days of gestation. (A) Control lung, canalicular stage, shows primitive alveolar septa; (B) diaphragmatic hernia lung, pseudoglandular stage, shows condensed mesenchymal tissue with large glands lined by columnar epithelium. Haematoxylin and eosin staining, original magnification: 20x2.5.

 


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Figure 3. Surfactant protein-A immunohistochemical staining of fetal lungs at 30 days of gestation. (A) Control lung shows dark-stained type II cells lining the alveolar septa. (B) Diaphragmatic hernia lung: the type II cells appear less abundant and smaller in size. SP-A immunostaining by avidin-biotin method, haematoxylin counterstain; original magnification: 20x2.5.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For a long time, animal models have been used to study pulmonary hypoplasia related to CDH. Current interest in prenatal therapy has accentuated the need for an animal model large enough to make in-utero surgery possible. In the absence of such animals with spontaneously occurring CDH, the diaphragmatic defect is usually created by surgical means at a given time in pregnancy. The outcome is generally assessed at term. Little information exists on the evolution of pulmonary hypoplasia in such surgical animal models. This baseline information is necessary to estimate how well these models mimic the human disease, and whether any in-utero intervention can correct the pulmonary hypoplasia.

Lung development in mammalian species undergoes several distinct phases, including embryonic, pseudoglandular, canalicular, saccular, and alveolar (Pringle, 1986Go). During these phases the airways, acinar, and vascular structures appear, grow, and mature in a certain chronological order. It is assumed that once beyond a critical stage, development of bronchi or other special structures cannot occur. For example, in man, the bronchial tree reaches its normal number of dichotomous divisions at around week 16 of gestation, whereas alveolization begins at 30–32 weeks gestation to complete postnatally (Pringle, 1986Go).

Pulmonary hypoplasia related to CDH is characterized by a decreased LBWR and DNA content of the lungs, a lower number of generations of airways, thickened interalveolar septa and decreased complexity of the respiratory acinus (Areechon and Reid, 1963Go; Kitagawa et al., 1971Go; Askenazi and Perlman, 1979Go; Wigglesworth and Desai, 1981Go). The number of vascular generations is also decreased, and there is increased muscularity of the peripheral arteries (Areechon and Reid, 1963Go; Kitagawa et al., 1971Go; Naeye Shochat et al., 1976). In addition to morphological immaturity, the surfactant produced by type II pneumocytes has been found deficient by some (Glick et al., 1992Go; Wilcox et al., 1994Go; Papadakis et al., 1998Go), although others have disputed this finding (Kato et al., 1996Go). The reduced number of airway and vascular generations in CDH suggests that lung growth must have been affected prior to 16 weeks gestation, i.e. already in the embryonic or early pseudoglandular phase. This has led to the assumption that the lungs are affected directly and independently of the diaphragmatic defect, or even that pulmonary hypoplasia is the primary defect in CDH (Iritani, 1984Go; Kluth et al., 1995Go).

Rabbit pulmonary development mimics that of the human lung in the respect that alveolization starts already in utero but proceeds postnally (Kikkawa et al., 1968Go; Pringle, 1986Go). In addition, the short gestation and large litter size makes rabbits ideal subjects on which to perform properly controlled surgical studies at a reasonable financial and time investment. Ohi (Ohi et al., 1976Go) was the first to produce CDH in fetal rabbits, and noted that the lungs remained small and hypoplastic on gross examination, although histologically they had matured significantly from the original pseudoglandular phase. In 1994 CDH was created in rabbit fetuses on day 24–25 of gestation (Fauza et al., 1994Go). At term the number of alveoli and smaller bronchi and bronchioli were reduced in the ipsilateral lung. The arterial wall of the small arteries was thicker in both ipsi- and contralateral lungs in fetuses with CDH compared with normal controls.

In the present study, we were able to create a diaphragmatic defect in rabbit fetuses on day 23 of gestation, i.e. at the end of the pseudoglandular phase. Over time the CDH lungs had a progressively lower LBWR than those of controls. In fetuses with CDH the MTBD was higher and lacked the terminal decline observed in controls. Alveolar size, reflected in the LM, did not change, whereas LMW, a measure of the interalveolar septal thickness, was significantly greater in fetuses with CDH. The rise in type II pneumocyte density, normally observed towards term, was significantly and progressively delayed in fetuses with CDH. These findings are compatible with an ongoing retardation of both lung growth and maturation. They were present in both lungs, although more marked on the ipsilateral side, as in human CDH.

The mechanism by which diaphragmatic hernia causes pulmonary hypoplasia remains unknown. Mechanical factors are generally considered important in lung growth. CDH may act as a space-occupying lesion, or it may interfere with fetal breathing movements, the lack of which has been shown to cause pulmonary hypoplasia (Wigglesworth and Desai, 1979Go). The retardation of lung maturation, usually considered to be under systemic control, is more difficult to account for. It may point to some, as yet unknown, paracrine/autocrine mechanism, possibly via the liquid secreted by the lungs themselves (Papadakis et al., 1997Go). Mechanical factors may also play a role in the differentiation of pulmonary epithelial cells in addition to their role in lung growth (Soto et al., 1996Go; Joe et al., 1997Go)

The persistent nature of the effect of CDH on lung growth and maturation is an intriguing finding. It is in accordance with the theory that the consequences of CDH depend on the phase of the lung development. During the embryonic and pseudoglandular phase, the result is a decreased number of bronchial and vascular generations, while during later phases the formation of the respiratory airways, alveoli and eventually cell differentiation are affected. Recent evidence suggests that the phases of lung development may have a wider time span and overlap to a greater extent than previously believed (Burri, 1997Go). In that case, the mere presence of a CDH may explain the wide variability of the disease and the wide spectrum of pulmonary pathology found in these cases. An inherent shortcoming of the rabbit model, and indeed of all surgical models, is that the diaphragmatic defect is not present until relatively late, so that information regarding the cause and early pathogenesis cannot be obtained.

Extrapolations to the human situation should be made with caution, as events during the embryonic and early pseudoglandular phase of lung development may modify later events and therapeutic response. Nevertheless, in rabbit fetuses, a surgical CDH on day 23 of gestation reproduces most features of the human CDH-associated lung hypoplasia, including the delayed maturation. The persistence of the deleterious effect until term gives additional justification to studies on prenatal therapy in such a model. The present results establish a reference on which such studies in the rabbit model can be based.


    Acknowledgments
 
J.W., X.G. and K.V. are recipients of a fellowship of the Bijzonder Onderzoeksfonds of the KU Leuven (0T/96/24). H.Y. is recipient of a research fellowship of the Kumamoto University, Japan. E.G. is recipient of a research fellowship of the European Commission (Eurofoetus Biomed 2 project BMH4 CT972383) co-financed by the Flemish Government (COF 98/012). K.V. is co-financed by the Academy of Finland.


    Notes
 
8 To whom correspondence should be addressed at: Centre for Surgical Technologies Minderbroedersstraat 17, B-3000 Leuven, Belgium. E-mail: Jan.Deprest{at}uz.kuleuven.ac.be Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on March 3, 2000; accepted on August 10, 2000.





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