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From the Lung Biology Research Program,*
Hospital for
Sick Children Research Institute, Department of Pediatrics, University
of Toronto, Toronto, Ontario, Canada; and the Department of Pediatric
Surgery,
Sophia Children’s Hospital, Erasmus
University Medical Centre Rotterdam, Rotterdam, The Netherlands
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathogenetic events resulting in the diaphragmatic defect and pulmonary hypoplasia are unknown. Classically, the primary defect is believed to be located in the diaphragm. Abdominal organs that herniate through this defect will interfere with normal pulmonary development. This results in a secondary defect: pulmonary hypoplasia and abnormal pulmonary vascular development.6,7 However, since the introduction of the 2,4-dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH, an alternative hypothesis has been suggested.8,9 This animal model is based on the teratogenic effects of the herbicide Nitrofen. When administered in the right dosage and at the right time to pregnant rats and mice, Nitrofen interferes with development of the lungs and the diaphragm of the offspring.9-11 Studies with this animal model have suggested that CDH might be due to primary disturbance of pulmonary growth into the pleuroperitoneal canal, thereby disturbing the growth of the posthepatic mesenchymal plate, the main origin of the diaphragm.8 After exposure to Nitrofen, 100% of the litter has a variable amount of lung hypoplasia, whereas depending on animal strain and timing of Nitrofen administration, a smaller percentage, varying from 60 to 90% in rats and 40 to 60% in mice, has a diaphragmatic hernia as well.9-11 Observations that both the contralateral and ipsilateral lung are hypoplastic suggest that proper pulmonary development in CDH is already impaired before the failed closure of the diaphragm. On the ipsilateral side, growth of the lung is hampered at a later phase by the presence of abdominal organs in the thoracic cavity and eventually interference with fetal breathing movements.
We hypothesized that the lungs are the primary target organ in the pathogenesis of pulmonary hypoplasia in case of Nitrofen-induced CDH. This implies that the lungs have to be hypoplastic before defective closure of the diaphragm has occurred. To test this hypothesis, we investigated early pulmonary development in an in vitro modification of the Nitrofen model. We have set up a foregut/lung explant system in which it is possible to study lung formation and branching morphogenesis without the influence of normal or defective closure of the diaphragm. The direct effects of Nitrofen on early pulmonary development in vitro were investigated by culturing explants in the presence of Nitrofen. In addition, the effects of removal of physical constraint on branching morphogenesis after Nitrofen treatment in vivo were tested. We show herein that Nitrofen interferes with branching morphogenesis both in vitro and in vivo. In contrast, initial lung bud outgrowth is not disturbed. Epithelial cell differentiation is attenuated even before effects of a defective closure of the diaphragm could have occurred. Furthermore, the interplay between proliferation and apoptosis is disturbed in lungs exposed to Nitrofen and therefore contributes to pulmonary hypoplasia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female (200–250 g) and male (250–300 g) Wistar rats were obtained from Charles River (St. Constant, PQ). The animals were kept in a controlled light-dark cycle and food and water were supplied ad libitum. Rats were mated overnight and the finding of a sperm-positive vaginal smear was designated day 0 of gestation. At 11 and 13 days of gestation (term = 22 days of gestation), timed-pregnant rats were killed by cervical dislocation after a short exposure to diethyl ether to anesthetize them. The fetuses were delivered by Caesarian section using aseptic surgical techniques and kept in Hanks’ balanced salt solution (Gibco, Burlington, ON). All protocols were evaluated and approved by the Animal Care Committee of the Hospital for Sick Children.
Explant Cultures
Foreguts and lungs were cultured according to previously reported studies.12 In short, foreguts (at 11 days of gestation) and lungs (at 13 days of gestation) were harvested from fetal rats under a dissection microscope using microsurgical techniques. The foreguts and lungs were transferred to porous membrane inserts (4 µm pore size) from Millipore (Bedford, MA), and incubated in 4-well cell culture plates from Nunc (Intermed, Denmark). The membrane inserts were pre-soaked in MEM (Gibco) for 1 hour before the explants were placed on them. The explants were incubated as floating cultures in 200 µl Dulbecco’s modified Eagle’s medium, nutrient mixture F-12 (Gibco) supplemented with 100 µg/ml streptomycin, 100 units/ml penicillin, 0.25 mg/ml ascorbic acid and 10% (v/v) heat-inactivated fetal bovine serum (Gibco). Heat inactivation of the serum was required to support long-term culture. The explants were cultured at 37°C in 95% air and 5% CO2.
Nitrofen
The herbicide 2,4-dichloro-phenyl-p-nitrophenylether (Nitrofen)
was obtained from Rohm & Haas Co. (Philadelphia, PA). To induce
congenital diaphragmatic hernia and pulmonary hypoplasia in
vivo, 100 mg Nitrofen dissolved in 1 ml olive oil was administered
orally on day 9 of gestation. To investigate the effects of Nitrofen
in vitro, the explants were exposed during the first day of
culture to a concentration of Nitrofen, which is similar to that used
in vivo: 0.25 mg.200 µl-1 medium.
To mimic the half-life of Nitrofen in vivo, the explants
were exposed to half of this during the second day of
culture.13,14
During the remaining culture period the
explants were cultured in medium alone. Nontreated explants and
explants cultured in the presence of vehicle dimethyl sulfoxide (DMSO;
BDH, Toronto, ON) in the same concentration as was used to dissolve the
Nitrofen served as controls (Table 1)
. Because Nitrofen is extremely
toxic, all handling was done in a fume hood or laminar flow using
protective gear to prevent inhalation and contact with the skin.
Disposal of all waste products containing Nitrofen was done according
to local regulations of The Hospital for Sick Children and all
experimental procedures were approved by the department of Occupational
Health and Safety of the Hospital for Sick Children.
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During the entire period of culture, lung formation and branching morphogenesis was monitored daily by phase-contrast microscopy. At approximately the same time point each day, branching morphogenesis was assessed by manually counting the number of terminal buds, and the lung explants were photographed.
Tissue Preparation
After 8 days (foregut) or 4 and 6 days (lung) of culture, explants were removed from the inserts and fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 16 to 18 hours, dehydrated in a graded series of ethanol, cleared in xylene, and embedded in paraplast (Oxford Labware, St. Louis, MO). Sections 5 µm thick were cut and mounted on Superfrost slides (Fisher Scientific, Unionville, ON) and baked at 42°C for 16 to 18 hours.
In Situ Hybridization
After dewaxing and rehydrating, tissue sections were postfixed in 4% (w/v) paraformaldehyde in PBS and permeabilized with proteinase K (20 µg·ml-1 in 50 mmol/L Tris/HCl, 5 mmol/L EDTA, pH 8.0) for 15 minutes at room temperature. After another postfixation step in 4% (w/v) paraformaldehyde in PBS for 5 minutes, the sections were carbethoxylated by 0.1% (v/v) active diethylpyrocarbonate treatment twice for 15 minutes to reduce background by inactivating RNases in the sections. Subsequently, the sections were equilibrated in 5x SSC (NaCl 0.75 mol/L, sodium citrate 0.075 mol/L) for 15 minutes and prehybridized for 2 hours at 58°C in 50% (v/v) formamide, 5x SSC and 40 µg·ml-1 salmon sperm DNA. The sections were hybridized overnight at 58°C with digoxigenin (DIG)-labeled Surfactant protein-C (SP-C) and Clara cell secretory protein-10 (CC-10) RNA probes in the same hybridization mixture. Rat specific SP-C and CC-10 cDNA fragments (330 and 315 bases, respectively) were DIG-labeled according to a protocol provided by the manufacturer (Boehringer Mannheim, Montreal, PQ). The next day, sections were washed in 2x SSC for 30 minutes at room temperature, 1 hour at 58°C and in 0.1x SSC for 1 hour at 58°C. The DIG Nucleic Acid Detection Kit from Boehringer Mannheim was used for immunological detection of the hybridized probe. Unspecific labeling was removed in 95% ethanol and the sections were counterstained with methyl green for 1.5 minutes. After dehydration in a graded series of ethanol and xylene, the sections were mounted with coverslips using Permount (Fisher Scientific).
Immunohistochemistry
Sections were dewaxed in xylene after heating them at 60°C for 5 minutes and rehydrated in a graded series of ethanol. Subsequently, antigen retrieval was performed by boiling the sections in a 10 mmol/L sodium citrate solution, pH 6.0 for two periods of 5 minutes in a microwave at medium high. Between the boiling periods, the sections cooled down for 20 minutes. After rinsing the slides in PBS endogenous peroxidase activity was blocked by exposing the slides to a 3% (v/v) hydrogen peroxide in methanol solution for a period of 10 minutes. After two rinses in PBS, the sections were blocked with 5% (v/v) normal goat serum and 1% (w/v) bovine serum albumin in PBS for the period of 1 hour. The sections were incubated overnight at 4°C with 1:1000 diluted primary monoclonal antibody to proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA). The sections were washed in PBS containing 0.05% (v/v) Tween followed by two washes in PBS alone. The sections were then incubated with a 200-fold dilution of biotinylated anti-mouse IgG for 1 hour at room temperature. After washing, the sections were incubated with avidin-biotin peroxidase complex (Vectastain) kit from Vector Laboratories (Burlingame, CA) for 2 hours at room temperature. Subsequently, after washes in PBS and Tris-buffered saline (TBS), the sections were developed using 3,3'-diaminobenzidine as substrate. Following washes in TBS and PBS, sections were counterstained with Carazzi’s hematoxylin. Subsequently, the sections were dehydrated in a graded series of ethanol and xylene and mounted with coverslips using Permount.
Terminal Deoxyribonucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) Assay
After dewaxing and rehydrating, tissue sections were incubated with proteinase K, 20 µg·ml-1 in 10 mmol/L Tris/HCl, pH 7.6 for 15 minutes at 37°C. Following two washes with PBS, the sections were incubated with 25 µl TUNEL reaction mixture (Boehringer Mannheim) for 1 hour at 37°C. Subsequently, the sections were washed in PBS and mounted with coverslips using Vectashield mounting medium with DAPI from Vector Laboratories (Burlingame, CA).
Data Presentation
All results are expressed as mean ± SE. Statistical significance was determined by one way analysis of variance followed by assessment of differences using a Tukey test for pairwise multiple comparison procedures.15 Linear regression was performed after transformation of the curves in straight lines by changing the numerical y axis scaling into a natural log scale. The rate of branching (slope) was depicted by the coefficient ± SE and differences were assessed using Student’s t-test. Significance was defined as P < 0.05.
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Results |
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After 72 hours of culture, lung formation was observed in all
foregut explants as a diverticulum arising from the foregut to form two
primary lung buds (Figure 1, B, F, and J)
. The timing of lung bud appearance was approximately the same in all
explants. The same holds true for initial lung bud outgrowth after
Nitrofen treatment in vivo, since the endodermal part of the
lungs in this group appeared similar at the moment of isolation (Figure 2D)
. However, the mesenchymal parts of
the lungs treated with Nitrofen in vivo appeared much
thicker than the mesenchymal parts of the control lungs (Figure 2D)
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During the remaining period of culture, dichotomous branching was
observed in all lung explants (Figure 2, E
-H). In the Nitrofen-exposed
explants, only lobular branching was observed (Figure 2, G and H)
, and
the rate of branching was reduced (Figure 3
and Table 2
). The
explants exposed to Nitrofen in vitro showed an arrest in
branching during the first 2 days of culture, the period of exposure to
Nitrofen. However, the rate of branching was also reduced during the
subsequent 2-day culture period (Figure 3)
. The number of branches of
explants exposed to Nitrofen in vitro was significantly
reduced when compared to control explants (P <
0.05; Table 2
). Exposure to Nitrofen in vivo did not result
in a statistically significantly reduced number of branches. When only
the 75% lowest mean numbers of branches were used for comparison,
statistical significance was reached (Table 2)
. No significant
differences were observed between control and DMSO-exposed explants.
The size of the explants was reduced after exposure to Nitrofen,
suggesting lung hypoplasia. Nitrofen exposure in vitro
resulted in more severe reduction in branching and size than exposure
in vivo.
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In the sections of the control explants and DMSO explants, major
airways and a pseudoglandular aspect were distinguished (Figure 4, A, B, E, and F)
. In the sections of
Nitrofen exposed explants, large air spaces, due to lobular branching
were observed (Figure 4, C, D, G, and H)
. After 4 days of culture, SP-C
mRNA was present in the control and DMSO exposed explants as well as in
the explants exposed to Nitrofen in vivo (Figure 4, A, B, and D)
. Expression was observed in the epithelial lining of the distal
airways. In contrast, no expression was observed in the explants
exposed to Nitrofen in vitro (Figure 4C)
. No differences in
the spatial expression pattern of SP-C mRNA were observed between DMSO,
control and Nitrofen in vivo treated explants. After 6 days
of culture, the same expression pattern was observed in the explants
exposed to Nitrofen in vitro (results not shown). After 4
days of culture, CC-10 mRNA expression was only observed in the DMSO
and control explants in the epithelial lining of the major airways
(Figure 4, E and F)
. This time, expression was not observed in any of
the explants exposed to Nitrofen (Figure 4, G and H)
.
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To investigate whether the interplay between proliferation and
apoptosis played a role in the early pathogenesis of Nitrofen-induced
pulmonary hypoplasia in vitro, we investigated
proliferation, using immunohistochemistry with an antibody against
PCNA.16
Apoptotic cells were detected using TUNEL
assay.17
Proliferating cells (PCNA-positive nuclei) were
localized mainly in the epithelial lining of the branching terminal
lung buds (Figure 5, A and B)
. A smaller
number of proliferating cells was observed in the mesenchyme (Figure 5, A and B)
. In mesenchyme of the Nitrofen-exposed explants, attenuated
PCNA immunoreactivity was observed (Figure 5C)
. Unexpectedly, in the
epithelial lining of the terminal buds, PCNA protein was localized
perinuclearly and in the cytoplasm, and not in the nuclei (Figure 5C)
.
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-F). All
nuclei of the cells were shown by staining with DAPI (Figure 5, G
-I). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We report here that Nitrofen exposure both in vitro and in vivo reduces branching morphogenesis before the time that closure of the diaphragm would normally occur in the rat (day 17 of gestation). Only the later phases of branching morphogenesis (ie, dichotomous branching) are affected, since initial lung bud outgrowth (ie, primary monopodial branching) and subsequent formation of the lobar bronchi (ie, secondary branching) were not affected in the Nitrofen-treated explants. The timing and degree of interference with branching morphogenesis by Nitrofen are in accordance with case reports of CDH in which the number of airway generations were assessed by the group of Lynne Reid.19,20 Both studies report a reduction in airway generations to about half the normal number. Because development of the bronchial tree is normally complete by the 16th week of intrauterine life, timing of the insult in affected human babies should be early in gestation.
Although in vivo exposure to Nitrofen at first did not result in a statistically significant reduction in the number of branches, the statistical significance was observed when the lower 75% of the lungs with reduced branching were considered from among all lungs evaluated. In a previous study, we have shown that in our hands administration of Nitrofen results in a rate of up to 80% diaphragmatic defects.21 Therefore, the lower 75% of the lungs with reduced branching might correspond to the most severe cases of pulmonary hypoplasia. All these data indicate that Nitrofen has a direct effect on branching morphogenesis and Nitrofen treatment might thus result in hypoplastic lungs before a diaphragmatic hernia occurs during development. This implies that in case of Nitrofen-induced CDH, the existing hypoplastic lungs might even induce the diaphragmatic defect, which has been suggested before by Iritani8 and by Cilley et al.9 However, results of the study of Iritani were based on experiments performed in the presence of Nitrofen during a period from day 5 of gestation until the day of sacrifice. In addition, both studies were done in mice, and the effects of Nitrofen were investigated in an in vivo situation, where influences of aberrant diaphragm development cannot be ruled out. To our knowledge, our study is the first to investigate the effects of Nitrofen on lung branching in vitro and after a limited period of exposure to Nitrofen, which is similar to the exposure of Nitrofen in the well established in vivo animal model.
In addition to the effects of Nitrofen on branching morphogenesis, the epithelial cell differentiation was also disturbed. Again, the effects of Nitrofen exposure in vitro were more severe than exposure in vivo, in that SP-C mRNA expression was not observed in the explants exposed to Nitrofen in vitro after 4 days, but only after 6 days of culture. In the Nitrofen-treated explants no CC-10 mRNA expression could be observed. Thus, in vitro treatment specifically resulted in a delay of epithelial cell differentiation. Whereas apoptotic processes did not change when evaluated by the number and localization of TUNEL-positive cells, PCNA immunoreactivity was clearly reduced in the mesenchymal component of the Nitrofen-treated explants. In addition, PCNA reactivity was only observed perinuclear and in the cytoplasm of epithelial cells of the explants exposed to Nitrofen. This unexpected result clearly indicated that proliferation is disturbed in explants exposed to Nitrofen. Consequently, a disturbed interplay between cell proliferation and apoptosis may be contributing to the observed hypoplasia in the lung explants exposed to Nitrofen.
Taken together, these data suggest that the primary defect in Nitrofen-induced CDH is located in the lungs and that the lungs are already hypoplastic before the formation of a diaphragmatic defect in the rat model. Our results corroborate with those of Cilley et al,9 who demonstrated in their murine model that Nitrofen-induced pulmonary hypoplasia exists with or without diaphragmatic hernia. The asymmetry of both pulmonary hypoplasia and the diaphragmatic defect can be explained by the fact that there is a difference in timing of closure of the diaphragms. The right diaphragm closes a little earlier than the left.22 It might well be that timing of the insult resulting in hypoplastic lungs and a diaphragmatic defect is crucial and consequently that it occurs more often on the left than on the right side. Strangely, administration of Nitrofen later during gestation results in a higher percentage of diaphragmatic defects occurring on the right side.10,11 In addition, the right lung is much bigger and consists of more lobes (4 in the rat and 3 in the human) than the left lung (1 in the rat and 2 in the human). This might result in a right lung providing a more sufficient basis for communication between the lungs and the diaphragm when the lungs grow into the pleuroperitoneal canal, which is disturbed in CDH as suggested by Iritani.8 Another possibility is that CDH should be considered as the result of a shared or a separate mechanism that affects, for instance, the mesenchyme of the lungs and diaphragm in a similar way. Although these problems are not clarified yet, it is clear that the occurrence of a diaphragmatic defect and pulmonary hypoplasia requires a sensitive and crucial interplay between several factors not yet identified.
Besides isolated or familial case reports describing chromosomal aberrations, until now a genetic origin or an etiological-environmental factor for CDH has been lacking.23 Therefore, the pathogenesis of pulmonary hypoplasia in the Nitrofen model of CDH should be extrapolated to the human situation with a fair amount of reserve. We postulate that the crucial event resulting in human CDH takes place during early branching morphogenesis in a susceptible genetic environment. There have been reports of families with a higher incidence of CDH, suggesting that a genetic defect is in part responsible for the etiology of the CDH cases.24,25 Therefore, more research efforts to unravel the genetic defects in this anomaly are justified and will lead to a better understanding of the pathogenesis of CDH.
Finally, we would like to postulate a new hypothesis describing the
pathogenesis of pulmonary hypoplasia in case of CDH: the dual-hit
hypothesis. In this hypothesis the pathogenesis of pulmonary hypoplasia
in case of diaphragmatic hernia is explained by two developmental
insults. The first insult occurs early on in development, before
diaphragm development, given a still unidentified background of genetic
and environmental factors. This insult affects both lungs during
branching morphogenesis in a similar fashion. After defective
development of the diaphragm, the second insult affects the
ipsilateral lung only at a later stage of development. In this
scenario, herniated abdominal organs will interfere with fetal
breathing movements of the ipsilateral lung, resulting in greater
impairment of the development of the ipsilateral lung than that of the
contralateral lung (Figure 6)
.
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Acknowledgements |
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Footnotes |
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Supported by a Medical Research Council of Canada Group Grant in Developmental Lung Biology, the David Vervat Foundation, and the Sophia Foundation for Medical Research.
Accepted for publication December 21, 1999.
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