www.elsevier.com/locate/jpedsurg

Journal of Pediatric Surgery (2007) 42, 1644–1651

Effect of antenatal tetrandrine administration onendothelin-1 and epidermal growth factor levels in thelungs of rats with experimental diaphragmatic herniaHan Lin, Yonggang Wang, Zhongxun Xiong, Yunman Tang, Wenying Liu⁎

Department of Pediatric Surgery, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, PR China

⁎ Corresponding author. Tel.: +86 28E-mail address: wenying@126.com

0022-3468/$ – see front matter © 2007doi:10.1016/j.jpedsurg.2007.05.017

AbstractPurpose: The aim of this study was to evaluate the effect of the traditional Chinese medicinetetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) andepidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatichernia (CDH).Methods: A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternallyadministered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH(n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered bycesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n =25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of eachfetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining wereperformed, and image analysis was performed after lung processing.Results: Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group wassignificantly lower than those of the Dex and EGF groups (P b .05). The lungs of fetuses with CDHshowed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast toimproved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statisticaldifferences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage ofmedial wall thickness, and vascular volume) were found (P b .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tetgroups (P b .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groupswas stronger than that in the control group (P b .05). There was no difference in lung EGF andET-1 immunoreactivity between CDH+Dex and CDH+Tet groups (P N .05).Conclusion: Antenatal treatment with Tet may improve lung growth and vascular remodeling, and itsmechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternallymay be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping toavoid the side effects of Dex.© 2007 Elsevier Inc. All rights reserved.

Index words:

Congenital diaphragmatichernia;

Endothelin;Epidermal growth factor;Tetrandrine

85422459.(W. Liu).

Elsevier Inc. All rights reserved.

Infants with congenital diaphragmatic hernia (CDH) havehigh perinatal morbidity and mortality despite recentadvances in perinatal care and improved understanding of

1645Effect of antenatal tetrandrine administraion

the pathophysiology of CDH. The main causes of mortalityin CDH are pulmonary hypoplasia and severe persistentpulmonary hypertension [1].

Regardless of experimental and clinical efforts usingmodalities directed toward reduction of pulmonary hyperten-sion and improved pulmonary gas exchange in infants withCDH [2-5], there has been relatively little impact on survivalof the most severely effected subset of infants with CDH [6].Antenatal therapies that promote lung growth before birthremain an appealing approach for fetuses with severe CDH.Fetal surgical intervention such as fetoscopic temporarytracheal occlusion is invasive, technically demanding, andlimited by the maternal and fetal risks [7,8]. In fact, evidencedemonstrated that tracheal occlusion did not improvesurvival or morbidity rates in human fetuses with CDHwhen compared with standard postnatal care [9]. Therefore,less invasive approaches such as antenatal pharmacologictreatment to stimulate lung growth and maturation have beenconsidered [10,11].

Tetrandrine (Tet), isolated from the root of Stephaniatetrandra S Moore, belongs to the bisbenzylisoquinolinealkaloids, which possess antioxidant and antifibrogeneticactivities. Tet has long been used in traditional Chinesemedicine as an antihypertensive and antiasthmatic agent [12].Liu et al [13] demonstrated, in the nitrofen rat model, that Tetimproved pulmonary vascular structural remodeling. How-ever, the effect of Tet on improving pulmonary hypoplasia inthe nitrofen-induced CDH rat model remains uncertain.

The aim of the present study was to evaluate the effect ofTet in improving CDH-related pulmonary hypoplasia andpulmonary hypertension and to explore the possiblemechanism of Tet administration related to epidermal growthfactor (EGF) and endothelin-1 (ET-1), which play animportant role in alteration of pulmonary growth andvasculature in this rat model.

ig. 1 Medial thickness of pulmonary artery and the RACethod in CDH lungs. A, RAC in a fetal rat lung. B, Percentage ofall thickness was calculated with the formula: [(ED − internaliameter)/ED] × 100 (H&E, original magnification ×400).

1. Materials and methods

1.1. Experimental design and animal model

All animals were approved and provided by the AnimalCare Committee of Sichuan University (Chengdu, China).Twenty adult female Sprague-Dawley rats with body weightsranging between 230 and 270 g (average, 250 g) were used.

Rats were bred after overnight controlled mating. Thevaginal smear method was used to detect pregnancy(gestational day 0, Gd 0). CDH was induced by maternaladministration of a single oral dose (100 mg intragastrically)of nitrofen (99% purity, Zhejiang Chemicals, Ningbo,Zhejiang, China) dissolved in 1 mL of olive oil on Gd 9.5.Control animals received an equal volume of olive oil alone.On Gd 18.5, nitrofen-fed pregnant rats were dividedrandomly into 3 groups: CDH, dexamethasone (Dex), andTet. Therefore, 4 groups of pregnant rats were designed:control (n = 5), CDH (n = 5), CDH+Dex (n = 5), and

CDH+Tet (n = 5). Dexamethasone (0.25 mg/kg) was givenby maternal intraperitoneal injection, and Tet was adminis-tered intragastrically. Cesarean delivery was performed onGd 21 (term, 22 days), and the fetuses were harvested.Under a dissecting stereomicroscope, the lungs of thefetuses were removed and the bilateral diaphragms werecarefully examined for CDH. The body weight (BW) andtotal lung weight (LW) of all fetuses with CDH were

Fmwd

1646 H. Lin et al.

recorded. Hernia size was estimated as percentage of totalthoracic content. For the fetuses with CDH, the lungs wereremoved and processed for histologic analysis.

1.2. Lung lavage and processing

After the mainstem bronchial stump was oversewn, thelungs were perfused via cannulation of the mainstembronchus with 10% formaldehyde at a pressure of 25 cmH2O for 48 hours, after which 2- to 3-mm-thick randomtransverse 5-mm-long slices of lower lobes were taken andembedded in paraffin. Sections 4 to 5 μm thick of theparaffin-embedded lung were stained with H&E andimmunohistochemical analyis was performed.

1.3. Lung and small pulmonary arterymorphometric study

1.3.1. Method of lung maturation measurementLung growth was represented by lung weight to body

weight (LW/BW) ratio. Morphometric evaluation of lungstructural maturation was performed by using the followingparameters: (1) The radial alveolar count (RAC), as an indexof alveolar proliferation and architectural maturity, originallydescribed by Emery and Mithal, was determined by countingthe number of airspaces lying on a line drawn perpendicu-larly from the center of a terminal or respiratory bronchiole tothe closest edge of the acinus (pleural or lobular connectivetissue septum) (Fig. 1A, ×40 magnification). The RAC wasused as a measure of the development of the terminalrespiratory unit. (2) Percentage of lung alveolar area per unitarea was measured by image analysis (Nikon, Tokyo, Japan).

1.3.2. Pulmonary vascular morphologyThe fetus and relevant organs were weighed, and the

lungs were subsequently immersed in 10% neutral bufferedformalin solution and embedded in paraffin. Paraffin-embedded lungs were cut into 5-μm sections, stained withH&E, and assessed using image analysis software (GT-4model, University of Chengdu Electronic Science, Chengdu,China) to delineate vascular morphology.

Table 1 Body and lung weights, lung morphometric analysis, and pu

Group

Control (n = 38) CDH (n = 25)

% LW/BW 4.15 ± 0.46 2.46 ± 0.51#

RAC 5.9 ± 0.7 2.4 ± 0.3#

% Alveoli 74.4 ± 11.6 47.5 ± 9.5#

ED 22.3 ± 12.9 24.8 ± 7.9% MT 11.38 ± 4.25† 16.4 ± 5.23Vv 0.37 ± 0.07† 0.20 ± 0.28

% LW/BW, RAC, and alveoli %: *P b .01 vs CDH; P b .05 vs controls; #P b .0ED: P N .05 among 4 groups.% MT and Vv: †P b .05 vs CDH.

Measurements of vessel wall thickness were performed byan observer blinded to the identity of the histology slides.Measurements of wall thickness were performed on smallpulmonary arteries (20-60 μm) associated with terminalbronchioles and distal airspaceswith an image analysis software(GT-4, University of Chengdu Electronic Science). Internaldiameter (ID) and external diameter (ED) were measureddirectly and expressed as the percentage of medial wallthickness (% MT), which was calculated with the followingformula: [(ED − internal diameter)/ED] × 100 [14] (Fig. 1B).

1.4. Immunohistochemistry and vesselvolume density

Immunohistochemistry for factor VIII was performed toassess vascular density. Paraffin-embedded slides fromformalin-fixed tissue were deparaffinized in CitriSolv (FisherScientific, Pittsburgh, PA). The sections were rehydrated byserial immersions in 100% ethanol, 95% ethanol, 70%ethanol, and water. Sections were digested with proteinaseK at a concentration of 500 g/mL for 10 minutes at roomtemperature and then washed with phosphate-buffered saline(PBS) with 2.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 138mmol/L NaCl, and 8.1 mmol/L KH2PO4. Endogenousperoxidase activity was reduced by immersion in 3%hydrogen peroxide in methanol. After rinsing, sections werecovered in 10% goat serum for 30 minutes and incubated withrabbit anti–factor VIII antibody (1:100) (Zhongshan, Beijing,China) diluted in PBS with 1% bovine serum albumin and0.1% sodium azide for 60 minutes. After incubation, thesections were rinsed with PBS and incubated with biotin-labeled secondary antibody diluted 1:200 in PBSwith 2%goatserum for 30 minutes. After incubation with the secondaryantibody, the sections were rinsed with PBS, incubated inavidin-biotin complex (Vector Laboratories, Burlingame, CA)for 30 minutes at room temperature, rinsed in PBS, anddeveloped with diaminobenzidine and hydrogen peroxide.The slides were lightly counterstained with hematoxylin, thendehydrated by sequential immersion in 70% ethanol, 95%ethanol, 100% ethanol, and CitriSolv before applying cover-slips. PBS was used as a negative control, substituting for

lmonary vascular remodeling

CDH+Dex (n = 21) CDH+Tet (n = 22)

3.78 ± 0.89* 3.65 ± 0.78*4.4 ± 0.4* 4.6 ± 0.5*

60.8 ± 7.8* 64.8 ± 8.6*23.6 ± 6.4 21.7 ± 5.612.11 ± 4.32† 11.83 ± 5.3†

0.34 ± 0.11† 0.32 ± 0.18†

1 vs controls.

Fig. 2 Lungs from CDH animals (B) are characteristic of the fetal canalicular stage, showing poorly formed saccules and thickened septalwalls compared with lungs of control rats (A), which show well-differentiated saccules and thin septal walls. Striking changes are seen in theCDH+Dex (C) and CDH+Tet (D) groups, showing an increase in air saccule size, thin septal walls, and maturation of the pulmonaryinterstitium (H&E, original magnification ×200).

1647Effect of antenatal tetrandrine administraion

primary antibodies. Immunostaining for EGF and ET-1protein was also performed by similar methods.

Five lung sections were selected and captured by digitalcamera for analysis. A point-counting method, in which thelung parenchymal tissue served as the volume of reference,was used to determine the volume fraction of factor VIIIimmunoreactive sites. A grid of 100 points is superimposedon color photographs captured by digital camera at amagnification of ×100.The number of points falling onimmunoreactive sites and on lung parenchyma wasrecorded. The vascular volume (Vv) (positive stained pro-file/parenchyma) was calculated as the ratio of the numberof points falling on factor VIII immunoreactive sites topoints on lung parenchyma.

Lung immunohistochemical staining in each group wasscored in a blinded manner. Four areas (magnification ×200)in each slide were selected blindly, and the intensity ofstaining was scored with respect to each histologic part.

1.5. Statistics

All quantitative data are expressed as mean ± SD. P b .05was considered statistically significant. Results from the 4groups of animals were analyzed by Student-Newman-Keulstest and Wilcoxon signed rank test using SPSS version 12.0statistical software (SPSS, Chicago, Ill).

Fig. 3 The medial thickness of the pulmonary artery is significantly incStriking changes are seen in the CDH+Dex (C) and CDH+Tet (D) groupgroup (B) (H&E, original magnification ×200).

2. Results

2.1. Body and lung weights, lung morphometricanalysis, and pulmonary vascular remodeling

Thirty-eight fetuses were harvested from the pregnantcontrol rats, and no fetus with CDH was found. CDH wasobserved in 68 of the 128 rat fetuses (53.1%) among theother 3 groups. For the experimental groups, only the fetuseswith CDH were included. Accordingly, the fetuses were di-vided into 4 groups: controls (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22) (Table 1).

Similar to that previously described in the literature, lungsfrom the fetuses with CDH of our experimental model weremarkedly hypoplastic, as evidenced by the LW/BW ratio,which was considerably lower than that of controls, and thedecreased RAC and percentage of lung alveolar area (%alveoli). Treatment with Dex and Tet resulted in fetal lungsthat were intermediate in size with a % LW/BWof 3.78% and3.65%, respectively, which were significantly larger than thatof the CDH group (2.46%, P b .01) but still somewhatsmaller than that of controls (4.15%, P b .05). Although theCDH+Tet group did not have fetal lungs comparable to thesize of the controls', these fetal lungs were structurally nearlymature as indicated by increased RAC and % alveoli whencompared with the immature lungs of the CDH group,

reased in the CDH group (B) compared with the control group (A).s, showing a decrease in medial thickness compared with the CDH

Table 2 Changes in EGF and ET-1 expression in fetal lung ongestational day 21

Group n EGF ET-1

Control ⁎ 38 59.69 ± 8.45 10.58 ± 1.47CDH 25 88.09 ± 5.68 25.36 ± 2.31CDH+Dex † 21 70.76 ± 4.82 16.16 ± 1.97CDH+Tet † 22 73.20 ± 4.76 15.89 ± 3.55

⁎ P b .01 vs controls.† P b .01 vs CDH.

1648 H. Lin et al.

although the herniated viscera were still present in the chestcavity at microdissection (Table 1, Fig. 2).

Comparison of the lungs between the CDH group andother groups showed that ED was not statistically different(P N .05), whereas % MTwas significantly different. Fetusesof the CDH group had a significantly increased % MTcompared with those of controls (P b .05), whereas CDH+Dex and CDH+Tet animals showed a significantly reduced% MT compared with CDH animals (P b .05). In addition tothe morphometric assessment of alveolarization, measure-

Fig. 4 Immunohistochemical staining of EGF in lung sections from the cTet group (D). Positive staining of EGF was detected in the bronchiolar eextima of vessels. Immunoreactivity of EGF is stronger in the CDH group (group (D). In addition, EGF immunostaining of the CDH+Dex group (C)was no difference in EGF immunoreactivity between CDH+Dex (C) and

ments of vessel volume density demonstrated increased Vvin the control, CDH+Dex, and CDH+Tet groups comparedwith the CDH groups (P b .05) (Table 1, Fig. 3).

2.2. Immunohistochemical expression of EGF andET-1 in rat fetal lungs

The predominant patterns of EGF and ET-1 staining in thefetal lungs were cytoplasmic and membranous. EGFimmunoreactivity was detected in the bronchiolar epithe-lium, part of the pulmonary vascular smooth muscle cells,and extima of vessels. The bronchiolar epithelium in CDHrat lungs demonstrated stronger EGF immunoreactivity thanthat of the control, CDH+Dex, and CDH+Tet groups(P b .01). In addition, EGF expression of the CDH+Dexand CDH+Tet groups was stronger than that of the controlgroup (P b .05). There was no significant difference in EGFimmunoreactivity in the lung between CDH+Dex and CDH+Tet groups (P N .05) (Table 2, Fig. 4). The positively stainedET-1 cells were localized in the bronchial epithelium,bronchial smooth muscle, pulmonary vascular endothelium,

ontrol group (A), CDH group (B), CDH+Dex group (C), and CDH+pithelium, part of the pulmonary vascular smooth muscle cells, andB) than in the control group (A), CDH+Dex group (C), and CDH+Tetand CDH+Tet group (D) was stronger than the control group. ThereCDH+Tet group lungs (D) (original magnification ×400).

Fig. 5 Immunohistochemical staining of ET-1 in fetal lung tissue sections from the control (A), CDH (B), CDH+Dex (C), and CDH+Tet (D)groups. ET-1 positive staining is seen in the bronchial epithelium, bronchial smooth muscle, pulmonary vascular endothelium, smooth musclein pulmonary arteries, and alveolar epithelium. Immunohistochemical staining is stronger in the CDH group (B) than the control (A), CDH+Dex (C), and CDH+Tet group (D). In addition, ET-1 immunostaining of the CDH+Dex group (C) and CDH+Tet group(D) were stronger thancontrol group. There was no difference in ET-1 immunoreactivity between CDH+Dex group (C) and CDH+Tet group lungs (D) (originalmagnification ×200).

1649Effect of antenatal tetrandrine administraion

smooth muscle in pulmonary arteries, and alveolar epithe-lium. Similar to EGF expression, ET-1 expression in theCDH group was stronger than that in the control, CDH+Dex,and CDH+Tet groups (P b .01), and ET-1 expression in theCDH+Dex and CDH+Tet groups was stronger than that inthe control group (P b .05). There was no significantdifference in ET-1 immunoreactivity in the lung betweenCDH+Dex and CDH+Tet groups (P N .05) (Table 2, Fig. 5).

3. Discussion

Congenital diaphragmatic hernia has an unacceptablemortality attributed to associated pulmonary hypoplasia andpulmonary vascular hypertension. Even sophisticated treat-ment techniques such as high frequency oscillatory ventila-tion (HFOV), extracorporeal membrane oxygenation, andfetal surgery have not significantly influenced the mortalityrate of patients with CDH. In an effort to reduce mortality, inutero correction has been proposed, such as prenatal

pharmacologic intervention to improve pulmonary maturity,and abnormal pulmonary vascular remodeling. In the nitrofenmodel, prenatal glucocorticoid therapy has been found tocorrect pulmonary immaturity [15], improve lung compliance[16], improve lung histology [17], and increase surfactantprotein B messenger RNA [18] and protein synthesis [19].Although the consequence of offering glucocorticoids has notbeen demonstrated, the potential side effects of antenatalglucocorticoid treatments, such as intrauterine growthretardation and adrenal suppression, to women and babiesshould be considered with the greatest caution [20]. There-fore, we propose another pharmacologic antenatal interven-tion, tetrandrine, which has fewer side effects, to evaluate itseffects compared with Dex and discuss its possible mechan-isms of action.

Tetrandrine, isolated from the root of S tetrandra SMoore,belongs to the bisbenzylisoquinoline alkaloids, and is re-cognized to possess antioxidant and antifibrogenetic activ-ities. Tetrandrine has long been used in traditional Chinesemedicine as an antihypertensive and antiasthmatic agent [12].

1650 H. Lin et al.

In the current study, we assess the beneficial effect ofadministration of Tet on the improvement of pulmonaryvascular structure, as evidenced by the % MT and Vvcompared with that of the CDH group (P b .05). In thiscontext, we propose that % MT and Vv could be used asindexes to measure the vessel wall thickness and vesselvolume density. In fact, this study confirms the findings ofLiu et al [13], who reported the effects of Tet on pulmonaryvascular structural remodeling in rats with CDH. Prenatal Tettherapy significantly reduced the percent medial thickness ofthe pre- or intraacinar arteries of CDH rats. It also increasedthe number of nonmuscularized arteries but decreased thenumber of fully or partially muscularized arteries inintraacinar vessels in rats with CDH. However, the mechan-ism of the effect of Tet on pulmonary vascular structuralremodeling of CDH rats is not completely understood.

The endothelium-derived vasoconstrictor ET-1 is a 21–amino acid peptide that is present in both vascular smoothmuscle cells and endothelial cells. ET-1 is present in theperinatal lung and is vasoactive in the fetus. In addition,increased ET-1 activity stimulates smooth muscle cellproliferation, which in turn increases pulmonary vascularresistance. Elevated expression and release of ET-1 have beendocumented in persistent pulmonary hypertension of thenewborn (PPHN), and Kobayashi and Puri [21] reportedelevation in ET-1 levels in CDH patients with PPHN.Furthermore, ET-1 is a mitogen for vascular smooth musclecells. Enhanced expression of ET-1 may also be attributed tothe abnormal pulmonary arterial muscularization in CDH. Inthis study, we found that the upregulated expression of ET-1in the CDH group strongly supports the reason for pulmonaryvasoconstriction and altered pulmonary vascular muscular-ization in CDH. We also showed that Tet decreased theexpression of ET-1. Therefore, we hypothesize that thebeneficial effect on CDH pulmonary vessel remodeling inthe CDH+Tet group is due in part to the decrease in ET-1activity partially antagonized by Tet.

More interestingly, in this current study, we also foundthat antenatal administration of Tet produced beneficialeffects of improved pulmonary hyperplasia as evidenced by% LW/BW, RAC, and % alveoli compared with those of theCDH group(P b .05). In this context, we propose that % LW/BW, RAC, and % alveoli can be used as direct indexes toevaluate the pulmonary growth. The mechanism of the effectof Tet on improving the pulmonary growth of rats with CDHhas not been reported. The results suggest that EGF might beinvolved in the therapeutic mechanism of Tet.

Epidermal growth factor plays an important role in celldifferentiation and development and accelerates lung devel-opment and maturation of hypoplastic fetal lungs in vitro[22]. In addition, some studies have demonstrated thatadministration of exogenous EGF improves lung growth onthe nitrofen-induced CDH model [23].

In the current study, the predominant difference betweenthe CDH hypoplastic lung and control lung was the strongEGF immunoreactivity in the bronchial and bronchiolar

epithelium in CDH lung (P b .05). The upregulatedexpression of EGF in the proximal airways in the CDHhypoplastic lung suggests persistence of the fetal stage ofpulmonary airway development in CDH, as demonstrated byreduced % LW/BW, RAC, and % alveoli in this study. Theseresults are in accord with previous reports from other authors[24]. EGF was probably elevated as compensation in CDHlungs to accelerate lung development. We also found that theexpression of EGF was decreased in the CDH+Tet group.Therefore, it is not clear whether the effect of Tet to improvelung hypoplasia is mediated by the decreased expression ofEGF, or the decreased expression of EGF may be the result ofpulmonary hypoplasia improved by Tet.

In summary, we found that nitrofen exposure in antenatalrats caused increased EGF and ET-1 expression andabnormalities in lung structure, including decreased vasculargrowth, abnormal vascular remodeling, and impairedalveolarization. We also noted that antenatal treatment withTet enhanced lung growth and improved vascular remodel-ing. Further studies are needed to determine the exactmechanisms of antenatal Tet administration in improvinglung structure, although the current findings suggest thatantenatal therapy with Tet might play a therapeutic role inimproving lung structure and seems to be involved in thedecrease in EGF and ET-1 expression. The presented resultssuggest that antenatal Tet administration might lead to ahopeful therapeutic option in the treatment of CDH and maybe effective in helping to avoid the side effects of Dex.However, this awaits evaluation by further experimental andclinical studies.

References

[1] Gosche JR, Islam S, Boulanger SC. Congenital diaphragmatic hernia:searching for answers. Am J Surg 2005;190:324-32.

[2] Hirschl RB, Philip WF, Glick L. A prospective, randomized pilot trialof perfluorocarbon-induced lung growth in newborns with congenitaldiaphragmatic hernia. J Pediatr Surg 2003;38:283-9.

[3] Boloker J, Bateman DA, Wung JT, et al. Congenital diaphragmatichernia in 120 infants treated consecutively with permissive hypercap-nea/spontaneous respiration/elective repair. J Pediatr Surg 2002;37:357-66.

[4] Stevens TP, Chess PR, McConnochie KM, et al. Survival in early andlate term infants with congenital diaphragmatic hernia treated withextracorporeal membrane oxygenation. Pediatrics 2002;110:590-6.

[5] Mann O, Huppertz C, Langwieler TE, et al. Effect of prenatalglucocorticoids and postnatal nitric oxide inhalation on survival ofnewborn rats with nitrofen-induced congenital diaphragmatic hernia.J Pediatr Surg 2002;37:730-4.

[6] Skari H, Bjornland K, Haugen G, et al. Congenital diaphragmatichernia: a meta-analysis of mortality factors. J Pediatr Surg2000;35:1187-97.

[7] Harrison MR, Sydorak RM, Farrell JA, et al. Fetoscopic temporarytracheal occlusion for congenital diaphragmatic hernia: prelude to arandomized, controlled trial. J Pediatr Surg 2003;38:1012-20.

[8] Harrion MR, Mychaliska GB, Albanese CT, et al. Correction ofcongenital diaphragmatic hernia in utero IX: fetuses with poorprognosis (liver herniation and low lung-to-head ratio) can be saved

1651Effect of antenatal tetrandrine administraion

by fetoscopic temporary tracheal occlusion. J Pediatr Surg1998;33:1017-22.

[9] Harrison MR, Keller RL, Hawgood SB, et al. A randomized trial offetal endoscopic tracheal occlusion for severe congenital diaphrag-matic hernia. N Engl J Med 2003;349:1916-24.

[10] Oue T, Taira Y, Shima H, et al. Effect of antenatal glucocorticoidadministration on insulin-like growth factor I and II levels inhypoplastic lung in nitrofen-induced congenital diaphragmatic herniain rats. Pediatr Surg Int 1999;15:175-9.

[11] Yu J, Gonzalez S, Diez-Pardo JA, et al. Effects of vitamin A onmalformations of neural-crest–controlled organs induced by nitrofenin rats. Pediatr Surg Int 2002;18:600-5.

[12] Achike FI, Kwan CY. Characterization of a novel tetrandrine inducedcontraction in rat tail artery. Acta Pharmacol Sin 2002;23:698-704.

[13] Liu W, Feng J, Jia H, et al. Effect of prenatal tetrandrine therapy onpulmonary vascular structural remodeling in the nitrofen-inducedCDH rat model. Chin Med J (Engl) 2000;113:813-6.

[14] Meyrick B, Reid LM. The effect of continued hypoxia on the ratpulmonary circulation. An ultrastructural study. Lab Invest1978;38:188-200.

[15] Suen HC, Bloch KD, Donahoe PK. Antenatal glucocorticoid correctspulmonary immaturity in experimentally induced congenital diaphrag-matic hernia in rats. Pediatr Res 1994;35:523-9.

[16] Losty PD, Suen HC, Manganaro T, et al. Prenatal hormonal therapyimproves pulmonary compliance in the nitrofen induced CDH ratmodel. J Pediatr Surg 1995;30:420-6.

[17] Ijsselstijn H, Pacheco BA, Albert A, et al. Prenatal hormones alterantioxidant enzymes and lung histology in rats with congenitaldiaphragmatic hernia. Am J Physiol 1997;272:L1059-65.

[18] Guarino N, Oue T, Shima H, et al. Antenatal dexamethasone enhancessurfactant protein synthesis in the hypoplastic lung of nitrofen-induceddiaphragmatic hernia in rats. J Pediatr Surg 2000;35:1468-73.

[19] Islam S, Cote GM, Hedrick HL, et al. Prenatal glucocorticoidsimprove surfactant protein B levels in fetal rat lungs with con-genital diaphragmatic hernia. Am J Respir Crit Care Med 1998;157:A559.

[20] Lacaze-Masmonteil T, Audibert F. Multiple courses of antenatalglucocorticoid treatment and fetal outcome. J Perinat Med2000;28:185-93.

[21] Kobayashi H, Puri P. Plasma endothelin levels in congenitaldiaphragmatic hernia. J Pediatr Surg 1994;29:1258-61.

[22] Zgleszewski SE, Cilley RE, Krummel TM, et al. Effects ofdexamethasone, growth factors, and tracheal ligation on the develop-ment of nitrofen-exposed hypoplastic murine fetal lungs in organculture. J Pediatr Surg 1999;34:1187-95.

[23] Li J, Hu T, Liu W, et al. Effect of epidermal growth factor onpulmonary hypoplasia in experimental diaphragmatic hernia. J PediatrSurg 2004;39:37-42.

[24] Guarino N, Solari V, Shima H, et al. Upregulated expression of EGFand TGF-alpha in the proximal respiratory epithelium in the humanhypoplastic lung in congenital diaphragmatic hernia. Pediatr Surg Int2004;19:755-9.