antenatal phosphodiesterase 4 inhibition restores...

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Antenatal phosphodiesterase 4 inhibition restores

postnatal growth and pulmonary development in a model

of chorioamnionitis in rabbits

L. Homer , E Launay , N. Joram , C. Jacqueline, P.-H. Jarreau , J Caillon,T

Moyon, B Branger, G Potel, JC Roze, C. Méhats , and C. Gras-Le Guen

LH : CHU Brest, Service de Gynécologie Obstétrique et Médecine de la Reproduction,

Brest, F- 29200, France ;

CJ, JC, CGL : Université de Nantes, Nantes Atlantique Universités, Thérapeutiques

Cliniques et Expérimentales des Infections, EA 3826, Faculté de Médecine, Nantes F -

44000, France ;

TM : a

Institut National de la Recherche Agronomique and University of Nantes, UMR-1280

Physiologie des Adaptations Nutritionnelles, CHU Hôtel Dieu, Place Alexis Ricordeau, HNB1,

44093 NANTES CEDEX 1, France (EU).

BB: Perinatal network “Pays de la Loire”, Nantes University Hospital,Nantes, France

JCR : National Institute of Health and Medical Research CIC004, Nantes University

Hospital,Nantes, France

NJ, EL, CGL : CHU de Nantes, Service de Néonatalogie, Hôpital Mère Enfant, 38

boulevard Jean Monnet, France Nantes cedex F - 44093, France ;

CM : Institut National de la Santé et de la Recherche Médicale (INSERM) U767, Paris,

France ;

PHJ : Université Paris Descartes, Faculté de Médecine, Paris, France ;

PHJ : CHU Port-Royal, Service de Néonatologie, 123 Boulevard de Port-Royal, Paris F-

75679 Cedex 14, France ;

JPET Fast Forward. Published on December 7, 2011 as DOI:10.1124/jpet.111.179085

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on December 7, 2011 as DOI: 10.1124/jpet.111.179085

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RUNNING TITLE PAGE

• Running title : antenatal PDEA inhibition improves pulmonary development

• Number of text page:20

• Number of tables: 3

• Number of figures: 4

• Number of references: 42

• Number of words:

• Abstract :242

• Introduction: 464

• Discussion: 1257

• Corresponding author:

Christèle GRAS – LE GUEN

Service de Néonatalogie - Hôpital Mère Enfant CHU de Nantes

38 boulevard Jean Monnet

Nantes cedex F - 44093, France

Tel : +33240083483 Fax : +33240083483

e-mail : [email protected]

• Liste of non standard abbreviations :

• PDE4: Phosphodiesterase 4

• BPD: Bronchopulmonary dysplasia

• BAL: Bronchoalveolar lavage fluid

• MIC : Minimum inhibitory concentration

• DMSO: Dimethyl sulfoxide • CFU : Colony forming units • HES: Hematoxylin-Eosin-Saffron

• Recommended section assignment: Gastrointestinal, Hepatic, Pulmonary and renal


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ABSTRACT 242words

Chorioamnionitis is implicated in the pathophysiology of bronchopulmonary disease, and the

associated inflammatory response is responsible for adverse effects on alveolar development.

The aim of this work was to analyze the effects of a phosphodiesterase 4 (PDE4) selective

inhibitor, rolipram (a modulator of the inflammatory response), in an experimental model of

chorioamnionitis on pulmonary development and on the processes of infection and

inflammation. Rabbit mothers were assigned to four groups: i) saline serum inoculation

(controls), ii) E. coli intra-uterine inoculation (C+), iii) rolipram infusion (R+), and iv) E. coli

inoculation + rolipram infusion (C+R+). High rates of morbility and mortality were noticed in

mothers and pups (5/13 pregnant rabbits in groups with rolipram). Alveolar development,

inflammation and infection were analyzed in pups at day 0 and day 5. At day 0, in the context

of chorioamnionitis, rolipram significantly decreased birth weight (p<0.01) relative to

controls (p<0.05). At day 5, weight normalized in the C+R+ group but not in group C+

relative to controls (p<0.001); moreover, alveolar airspace volume was preserved in group

C+R+ but not in group C+ animals (p<0.05). Interstitial volume decreased in group C+

animals versus controls (p<0.05) but was preserved in group C+R+. Specific alveolar area

was not significantly modified by rolipram. No significant difference was found concerning

bronchoalveolar lavage cellularity, and all blood cultures remained sterile. In this model of

impaired alveologenesis, rolipram significantly preserved specific alveolar density. However,

PDE4 inhibition induced antenatal fetal demise and growth retardation.


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INTRODUCTION 464 words

Bronchopulmonary dysplasia (BPD) remains a major concern in very premature

infants despite recent advances in perinatal care. BPD is characterized by the arrest of

secondary septation, previously described as an alveolar number decrease and an alveolar

enlargement, and is associated with thinned septa and minimal capillary development

(Kinsella et al; 2006). Alveolarization occurs postnatally in humans and premature, in utero

exogenous disturbances of immature lungs are thought to induce BPD through inflammatory

processes of the airway (Coalson JJ et al; 1999). Main aetiologies are the prematurity itself,

and also perinatal care such as hyperoxia and mechanical ventilation (Kraybill EN et al;

1989). Relationships between BPD and inflammation were established by Ogden et al., who

found increased inflammatory cell counts in bronchoalveolar lavage fluid (BAL) of neonates

exposed to hyperoxia or mechanical ventilation who subsequently developed BPD (Ogden BE

et al; 1983). Chorioamnionitis and neonatal infections have been implicated in the

development of pulmonary defects. Actually, correlation between inflammation, premature

delivery, and BPD has been demonstrated in humans (Kallapur SG et al; 2001) and animals

(Kallapur SG et al; 2004). Yoon, et al. have shown that high levels of IL-6, IL-1ß, IL-8, and

TNF-α found in amniotic fluid as early as 5 days before delivery was predictive of BPD in

premature neonates (Yoon BH et al; 1997). This observation was confirmed by Gomez et al.,

who showed that fetal morbidity correlated with an inflammatory response as a result of high

concentrations of IL-6 in fetal plasma, even in the case of asymptomatic chorioamnionitis

(Gomez R et al;1998).

As modulators of the inflammatory response, phosphodiesterase (PDE) inhibitors have

been widely studied in chronic pulmonary inflammation deseases (Hatzelmann A et al; 2010;

Giembycz MA; 2006). Among the 11 families of PDEs, selective inhibition of PDE4


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increases intracellular cAMP levels, particularly in inflammatory cells (T and B lymphocytes,

polymorphonuclear neutrophils), respiratory epithelial cells, and endothelial cells (Torphy TJ;

1998), thus modulating cellular trafficking and cytokine and chemokine responses (Bender

AT and Beavo JA; 2006; Sanz MJ et al; 2005). Moreover, inhibition of PDE4 limits

interstitial fibrosis and enhances muscular relaxation in airways (Kohyama T et al; 2002).

Recently, selective PDE4 inhibition was tested in a common murine postnatal hyperoxia

model of BPD. While de Visser, et al. found that PDE4 inhibition prolonged median survival

and reduced alveolar fibrin deposition and lung inflammation (de Visser YP et al; 2008),

Mehats et al. observed diminished weight gain without restoration of impaired alveolarization

induced by hyperoxia (Mehats C et al; 2008).

The aim of this study was to test the antenatal effects of the prototypical PDE4

inhibitor, rolipram, in a previously described model of chorioamnionitis-induced BPD in the

rabbit (Gras-Le Guen C et al; 2008). Pulmonary development was assessed by lung

morphometry. Inflammation and infection were determined by cellularity and culture of blood

and BAL.


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METHODS

Antenatal infection

Pregnant and newborn New Zealand rabbits were used in the study. The RS 218 (O18:K1:H7)

strain of Escherichia coli K1 was used and has been characterized for virulence factors and

invasion genes (Huang SH et al; 1995). The minimum inhibitory concentration (MIC) of this

strain for ceftriaxone was 0.06 mg/L. The intra-uterine inoculation protocol, as previously

described (Gras-Le Guen C et al; 2003), was approved by the Animal Care Committee of the

University of Nantes experimental therapy department (Nantes, France). A total of 25 female

New Zealand white rabbits weighing 3.7–4.5 kg (CEGAV, Saint Marc d’Egrenne, France)

were obtained 10 days before the end of their usual 31- to 32-day gestation period and housed

at the Nantes School of Medicine (Nantes, France) in appropriate cages with a special place

for nidation. The 25 female rabbits were provided water and food ad libitum consisting of

antibiotic-free granules. Animals were randomly assigned to four groups: i) controls (saline

serum inoculation), ii) C+ (E.coli intra uterine inoculation), iii) R+ (saline serum inoculation

+ rolipram infusion), iv) C+R+ (E.coli inoculation + rolipram infusion). Pregnant rabbits in

all groups (n = 25 for both groups) were treated with 200 mg/kg/day intravenous ceftriaxone

(Rocephine®; Roche, Meylan, France) via a catheter inserted into a marginal vein of the ear.

Treatment began 6 h after intrauterine inoculation and lasted 4 days, until spontaneous

delivery. Pregnant rabbits in all groups received dimethyl sulfoxide (DMSO), the rolipram

diluent. DMSO was injected alone (0.02 mg + 20 mL saline serum) or with rolipram (R6520,

Sigma-Aldrich, France), according to the experimental group. Posology of rolipram was 0.250

mg/kg in our study. Suspension of rolipram was done at 7.3 mg/mL in DMSO. The rolipram

treatment was started as the antibiotic treatment 6 h after intrauterine inoculation and lasted 4

days, until spontaneous delivery. For administration, rolipram was infused with 20mL of

0.9% saline serum during a 60 min continuous infusion. On day 28 of gestation, the animals


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were anesthetized with PROPOFOL LIPURO® 1% (B BRAUN PHARMA) and ketamine (25

mg/kg). Laparotomy was then performed via a 2-cm vertical midline incision below the

gravid uterus. After exposing the uterus, a single injection of E. coli (1×103 colony forming

units (CFU) in 1 mL of 0.9% NaCl solution) or sterile vehicle was performed under visual

control into one of the uterine horns facing a fetoplacental unit. Slight aspiration was

performed before injection to verify correct intra-amniotic positioning of the needle. The

incision was then closed in layers and the animals were returned to their cages.

After spontaneous birth, rabbit pups were suckled until euthanasia. They were

exsanguinated at birth (day 0) or on day 5, under deep anesthesia with intraperitoneal

Pentothal. Blood was obtained by cardiac puncture after animals failed to blink an eyelid or

move a leg muscle following local stimulation. Five to ten rabbit pups per group were used

for either bronchoalveolar lavage or morphometric study.

Assessment of lung morphometry

Lungs were gently extracted and fixed with 4% paraformaldehyde through a polyethylene

tracheal cannula at a constant pressure of 30 cm water for 10 minutes. The trachea was then

ligated, and the lung was immersed in 4% paraformaldehyde for 24 h. Lung volumes were

measured by the displacement method in the fixative solution (Scherle W, 1970). Both lungs

of each rabbit were paraffin-embedded and 4-m frontal sections were cut in the medial part of

the lungs, and stained with Hematoxylin-Eosin-Saffron (HES). All lung lobes were used. All

morphometric evaluations were performed by one observer (L.H.) blinded to group

assignment. A microscope (Leitz, Weszlar, Germany) connected to a television screen by a

video camera (Sony, Tokyo, Japan) was used. Volume densities of pulmonary parenchymal

structures (alveolar airspace, airways, blood vessels larger than 20 m in diameter, and


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interstitial tissues), and alveolar surface density were measured using the point counting and

mean linear intercept methods, as previously described (Weibel ER, 1997). Lightmicroscope

fields were quantified at an overall magnification of 400×, using a 42point, 21-line eyepiece

graticule placed on the television screen. Twenty fields (10 per lung) were evaluated per

animal by a systematic sampling method from a random starting point. To correct for

shrinkage associated with fixation and paraffin processing, area values were multiplied by

1.22, a factor calculated during a previous evaluation (Franco ML et al; 2002). All

morphometric data were expressed as relative and absolute values, as described by Burri and

coworkers (Burri PH et al; 1974). Relative values (volume density or surface density) were

those obtained directly from morphometric measurements of tissue sections. Absolute values

(total volume or surface area per lung) were determined by multiplying the relative values by

lung volume.

Evaluation of cell proliferation

Cell proliferation was assessed at day 0 by immunohistochemistry, using a monoclonal mouse

antibody against rat proliferating cell nuclear antigen as primary antibody, diluated 1:1500

(Mouse anti-rat PCNA (Clone PC-10), DakoCytomation, Ca# M0879). Peroxidase-linked

goat anti-mouse immunoglobulin (Santa Cruz Biotechnology, Cat# SC 2005) was used as

secondary antibody, diluted 1:1500 (Horseradish Peroxydase, HRP). The alveolar PCNA

index was determined by counting the immunostained nuclei per alveoli in 15 fields of

alveolar parenchyma per animal using a Quantimet Q550 (Leica Microsystem, Cambridge,

UK) (Namyslowski G et al; 2004) .

Evaluation of cell death


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Cell death was assessed at day 0 using the DNA specific dye Hoechst 33258 (Calbiochem,

Merck Biosciences, VWR, Fontenay-sous-Bois, France), at 1 mg/mL in Hank’s balanced salt

solution without phenol red on formalin-fixed paraffin sections. Sections were mounted with

Prolong Antifade medium (Molecular Probes, Eugene, OR, USA). Fluorescence was observed

with a fluorescence microscope (Axiovert 200-M; Carl Zeiss, Göttingen, Germany) equipped

with an ApoTome slider, which eliminates image blurring. Cells were visualized with a

63/1.4× oil immersion lens. Image processing was performed using an AxioCam MR CCD

camera and AxioVision 4.0 software (Carl Zeiss, Göttingen, Germany).

Evaluation of lung elastic fiber content

To evaluate lung parenchyma elastic fiber length content at day 5, lungs sections were stained

by the Weigert technique. The standard morphometric methods described above were then

used [i.e., counting the number of points touching alveolar parenchyma (point counting) and

the number of intersections between the graticule lines and the elastic fibers (mean linear

intercept)]. (Weibel ER, 1997)

Assessment of vascularization

Vascularization was assessed at day 5 by immunohistochemistry using a monoclonal antibody

against human CD31 (Clone JC/70A, DakoCytomation, Ca#M0823) as primary antibody,

diluted 1:50. The vascular development index was determined by counting the immunostained

nuclei per alveoli in 25 fields of alveolar parenchyma per animal, using a Quantimet Q550

(Leica Microsystem).

Assessment of total and specific PDE activity in pup lungs at day 0


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Whole lung tissues were homogenized in ice-cold hypotonic buffer, (100 mM Tris-HCl pH

7.4, 2 mM MgSO4, 2 mM EDTA, 50% glycerol, and 1 mM 2-β-mercaptoethanol

supplemented with P2714 protease inhibitor cocktail, Sigma) using an all-glass homogenizer.

Aliquots of homogenates were assayed for cAMP-PDE activity according to the method of

Thompson and Appleman (Thompson WJ et Appelman MM; 1971). PDE activities were

measured with 1 µM cAMP as substrate (GE Healthcare, Amersham, Buckinghamshire, UK).

PDE4 activity was defined as the fraction of cAMP PDE activity inhibited by 10 µM rolipram

as described previously (Schmitz T et al; 2007). Protein concentrations were determined using

the protein assay kit from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as

standard.

Assessment of inflammation: Broncho-alveolar lavage (BAL)

BAL was performed as previously described (Franco ML et al; 2002). Rabbits were

exsanguinated and the thorax was opened to expose the lungs and trachea. A short length of

tubing was inserted into the trachea and ligated, and then lungs were immediately lavaged.

BAL was performed at birth and at day 5 using a total of 1 mL sterile saline. BAL fluid was

centrifuged and total and differential cell counts were performed. Cytodifferentiation was

performed on cytospin preparations stained with Diff-Quick (Scientific Products, McGraw

Park, IN, USA).

Bacteriological analysis

Blood cultures

At the time of sacrifice, blood was withdrawn from the hearts of mothers and pups for

quantitative cultures; 1-mL blood samples were mixed with 900 mL of heparinized (5,000

IU/L) physiological serum. After centrifugation, 100 mL from each pellet were plated on MH


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agar (Difco; Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 37°C for 24 h

before performing bacterial counts. This method allows detection of an inoculum of 102

CFU/mL. (Gras-Le Guen C et al; 2008)

Pulmonary cultures

At the time of sacrifice, lung samples were homogenized in 100 µL of sterile saline solution

and diluted to 10-2 and 10-4 to prevent the possibility of carry-over. Next, 50 mL of each

dilution was spread on EMB plates using a spiral system plater (Interscience, BV, Louvain,

Belgium). After 48 h of incubation at 37°C, viable bacteria were counted.

Statistical analysis

Statistical analyses were performed with SPSS Software (version 15.0, SPSS Inc.

Chicago, IL USA). Multiple group comparisons were performed using a 2X2 factor ANOVA

analysis. Survival was evaluated by Kaplan-Meier survival function and the logrank test. A p-

value < 0.05 was considered to be statistically significant.


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RESULTS

Assessment of survival and growth at day 0 and day 5

Survival. Five pregnant rabbits died between inoculation (day 28 of gestation) and

delivery, three in group C+R+ (days 29, 29 and 31) and two in group R+ (both at day 31);

however, mortality rates did not significantly differ between the four groups. No mother died

after delivery.

Among pups, intrauterine death was significantly increased in groups C+R+ and R+

compared to controls (p<0.0001) and group C+ (p<0.03), and postnatal mortality was

significantly increased in groups C+ (p<0.03) and C+R+ (p<0.04) relative to Controls (Figure

1). There were significant differences in survival of live born pups at day 5 between controls

(97±2%) and groups C+, C+R+ and R+ (31±6, 22±8 and 40±5%, respectively, p<0.0001, by

Logrank)

Body-weight gain. (Table 1). Animals in Rolipram groups (R+ and C+R+) and

infection groups had significantly lower weight at birth. Five days after birth, infection and

Rolipram had no significant effect, but we observed a strong interaction between these two

factors (F1/22=44,6, p=0,000001). Weights were preserved between control animals and

C+R+ animals

All maternal and fetal blood and tissue cultures remained sterile. We previously demonstrated

in the same experimental model early fetal infection 8 h after bacterial challenge with 5 (29%)

out of 17 fetal blood positive cultures and 15 (88%) out of 17 placental positive cultures with

a mean quantitative value of 4.5±1.4 log10CFU.g-1 of tissue) (Gras-Le Guen C et al; 2008)

Lung morphometry at day 0 and day 5


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Examination of postnatal lung sections under light microscopy at day 5 showed that infected

rabbit lungs exhibited a diffuse and simplified lung structure with enlarged air spaces and

fewer secondary septa, whereas infected rabbit treated antenatally with rolipram exhibited an

alveolar morphology similar to control animals . Quantification by morphometric analysis

highlighted the following features (Tables 1 and 2):

-lung volume: At day 0, no difference was found between the four groups for absolute

lung volumes but we noticed a significant effect of Rolipram on specific lung volume

(F1/22 = 7,1; p=0,01). At day 5, absolute lung volume was significantly reduced in

infection groups (F1/21 = 7,6; p=0,01) with a strong interaction between infection and

rolipram effects (F1/21 = 52,3; p=0,000001), but we did not onserve any effect of

infection or Rolipram on specific lung volume.

-alveolar airspace (Vva): At day 0, we observed a significant effect of Rolipram on all

alveolar airspace parameters (volume density, absolute volume, specific volume).

Moreover, we noticed a strong interaction effect on volume density (F1/22 = 16,0;

p=0,0005). At day 5, we reported some significant interaction effects on all alveolar

airspace parameters. Rolipram seemed to have the same deleterious effect than

infection on alveolar airspace, but preserved alveolar volume when used in infected

animals.

-alveolar surface (Sva): At day 0, we did not observe any significant effect of

infection nor Rolipram on alveolar surface. At day 5, we reported a significant effect

of Rolipram on all alveolar surface parameters (area density, absolute area and specific

area).


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-interstitium (Vvi): At day 0, specific volume was significantly increased in Rolipram

groups (C+R+ and R+). At day 5, we observed an interaction effect on relative volume

density (F1/21 = 6,1; p=0,02).

-airways: At day 0, specific volume was significantly increased in Rolipram group,

and at day 5, we observed an interaction effect on absolute volume (F1/21 = 10,9;

p=0,003).

Blood vessels: At birth, specific volume was significantly increased in Rolipram group, with

an interaction effect on the 3 parameters. At day 5, we observed the same interaction effects

on the blood vessels. Once again, morphometric parameters were decreased in group C+ but

maintained in group C+R+ (table 2). Moreover, microvascularization studied by

immunohistochemistry labeling was significantly altered in all groups as compared to controls

(Figure 2A).

Evaluation of lung elastic fiber content

Morphological analysis of elastic fibers at day 5 showed a significant decrease in the

number of alveolar fibers in the pup’ lungs of groups C+, C+R+ and R+ versus controls [8±4,

16±5, 4±2, vs. 40±25, respectively, (p<0.05)]. The number of alveolar fibers tended to be less

decreased in group C+R+ than in groups C+ and R+, but the difference was not significant

(Figure 2B).

Assessment of apoptosis and cell proliferation

Apoptosis was present in the controls, and the group C+ parameters were not

associated with an increase in cell apoptosis (Figure 3A). A significant decrease was noticed


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in group R+ compared to both controls and C+R+ (p<0.001). For cell proliferation, an

increase was noticed in the C+ group relative to controls (p<0.05; Figure 3B).

Assessment of inflammation in BAL fluid at day 0 and day 5 and PDE4 activity in lung

at day 0

Mononuclear cell count in BAL fluid. No difference was found in the inflammatory

cell count in BAL fluid, either at day 0 or at day 5. However, group C+R+ animals showed a

trend, although not significant, to an increase of mononuclear inflow (Table 3).

PDE4 activity in lung. A global and equivalent AMPc-phosphodiesterase activity was

noticed in all conditions at day 0. Mean PDE4 specific activity represented 48.7±5.1% of the

global AMPc-phosphodiesterase activity expressed in the pups’ lung for all conditions, and

did not change subsequent to the experimental condition (Figure 4).


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DISCUSSION 1257 words

In the present study, we report that inhibition of PDE4s by rolipram in rabbit pups exposed to

chorioamnionitis preserved ante and postnatal alveolarization, without modifying the

inflammatory response. However, we observed marked intrauterine growth retardation and a

very high incidence of stillbirth in animals treated with rolipram, results not yet reported in

this model. Rolipram is the prototypical PDE4 selective inhibitor. PDEA 4 enzyme is the

main cAMP metabolising enzyme in immune and inflammatory cells, airway smooth muscle,

and pulmonary nerves; its inhibition suppresses the recruitment and activation of several

inflammatory cells (neutrophils, CD8 T cells, and macrophages) known to have a crucial role

in the pathophysiological processes of bronchopulmonary dysplasia (Hayes D et al; 2010;

Sanz MJ et al; 2005). In this context, we chose to test this new treatment in a previously

described model of antenatal infection with subsequent impaired alveolarization in the rabbit

(Gras-Le Guen C et al; 2008). The detection of AMPc-phosphodiesterase activity in our study

confirmed that PDE4s are expressed in pups’ lungs. The very short half-life of the molecule (

1.9 ±0.6 hour in the rabbit when using the 0.2 mg/kg dose) can probably explain the absence

of significant changes in the level of expression between the different groups, because the

assays were possible only 6 to 12 hours after birth, several hours after the last injection

(Krause W et al, 1988). However, we can observe a tendency to a decreased expression in the

infected + rolipram group (C+R+) when compared to the infected group (C+). The small

posology that we used (0.25 mg/kg/d) is possibly responsible for this lack of difference, but

was imposed by the poor tolerance to higher doses observed in this model. However, these

results confirm that PDE4 is a potential target in fetal and neonatal lungs.


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Antenatal infusion of selective PDE4s inhibitor preserves alveologenesis in the

context of chorioamnionitis. We observed conservation of specific interstitial, vascular,

airways and alveolar airspace volumes at day 0, and persistent effects at day 5 with some

significant interaction effects on alveolar airspace, interstitium, airways and vascular

morphometric parameters. Rolipram effects seem clearly different in infected or in non-

infected fetuses and newborns. These observations are consistent with the results of Woyda et

al., using a different PDE4 inhibitor (cilomast) in a different experimental model of hyperoxia

alveologenesis impairment in the mice (Woyda K et al; 2009). However, the lack of a control

group and the quality of morphometric determinations made the interpretation of the exact

effects of PDE4 inhibition on disturbed alveolarization difficult (Mehats C et al; 2009).

Mehats et al., in the same model in rat indicated that PDE4 inhibition with rolipram did not

enhance alveolarization in rat pups exposed to hyperoxia, and may directly affect

alveolarization in rat pups exposed to room air (Méhats C et al; 2008). The authors suggested

that this last point could be explained by the profound effect of PDE4 inhibition on pups’

weight gain that could interfere with normal alveolarization as much as antenatal infection

interfere (Gras-Le Guen C et al, 2008). In this infectious and inflammatory context, benefic

effects of PDE4 inhibition observed on alveologenesis in infected pups is possibly mediated

by some specific anti-inflammatory mechanisms whereas poor weight gain and poor

alveolarization both could reflect poor animals nutrition.

Rolipram infusion was also concomitant with intrauterine growth restriction. In fact,

this experimental model of antenatal infection has previously been associated with neonatal

growth alteration, but only with a postnatal onset (Gras-Le Guen C et al; 2008). In this present

work, antenatal growth restriction is noticed at birth in the two groups exposed to rolipram

versus the infected group (C+) and controls. However, postnatal growth restriction occurred

as early as 5 days after birth in the infected group (C+) but not in group C+R+ animals, where


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rolipram seemed to have preserved postnatal growth. However, de Visser et al. and Mehats et

al. observed a weight loss in rat pups exposed to rolipram in an experimental model of

hyperoxyia-altered alveolarization (de Visser YP et al; 2008; Méhats C et al; 2008). In

Humans, Fabbri et al. observed weight loss in the group of patients with chronic obstructive

pulmonary diseases treated with roflumilast compared to a control group (Fabbri LM et

al;2009 ). To explain the weight loss, one hypothesis was that pups treated with rolipram were

underfed because of side effects limiting the pregnant rabbits’ feeding. Rolipram is known to

have adverse effects on the central nervous system that accounts for nausea, vomiting, and

enhanced gastric acid secretions (Zeller E et al; 1984; Barnette MS et al; 1995). However,

mother rabbits did not lose weight during the delay from inoculation through delivery.

Secondly, rolipram on its own increases lipolysis as described in an in vitro model of rat pups

(Nakamura J et al; 2004; Snyder PB et al; 2005). In addition, toxicological reports during

preclinical studies demonstrated significant inflammation of the intestinal tract, suggesting

pathological absorption of food (Dagues N et al; 2007; Larson JL et al; 1996). This

mechanism would better explain the differences noticed between the C+ and control groups

and group C+ versus groups C+R and R+ at birth, because animals treated with rolipram

made up for the weight restriction as soon as rolipram infusion was stopped, and group C+R+

recovered normal weight at day 5 compared to controls.

Despite the fact that rolipram showed some protective effects on alveologenesis in the

chorioamnionitis model, high rates of morbidity and mortality were noticed in mothers and

pups. Such a mortality rate has not been reported with pregnant rabbits in this experimental

model –Gras-Le Guen C et al; 2008). Hemodynamic effets have been described on

anesthetized and ventilated animals (decrease in pulmonary arterial pressure and systemic

arterial pressure, increase in cardiac output ), without possibility to assess morbo-mortality

during these veru short-term experiences (Schermuly RT et al; 1999). Cardiac toxicity has


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been described in rabbits by Shahid and Rodger, who showed an in vitro increase in the

intracellular concentration of cAMP that led to chronotropic effects on atrial muscle fibers

(Shahid M and Rodger IW; 1989). Nevertheless, this effect did not induce cardiac arrhythmias

(Vaughan Williams EM; 1987). PDE4 inhibitors have been used in humans with a well-

described adverse event profile, and such severe or fatal effects have not been reported

(Fabbri LM et al; 2009). The fatal adverse events in the rabbit are probably specific to this

experimental model and constitute a limitation, as previously observed with corticotherapy

(Pratt L et al; 1999). In utero death of pups in groups C+R+ (52.1%) and R+ (46.8%) differed

significantly from both controls (0.7%) and group C+ (18.9%), and the increase in mortality

in group C+R+ compared to group C+ was probably related to the cumulative deleterious

effects of chorioamnionitis and rolipram infusion. We hypothesized that the tocolytic effect of

rolipram described by Schmitz et al. may have played a role in this high rate of in utero

mortality (Schmitz T et al; 2007); however, inoculation-delivery delay did not differ between

the four groups. We also suggested that peripartum maternal behavior associated with

maternal neglect could be implicated in the mortality of the pups post-delivery as an indirect

consequence of rolipram. Interestingly, de Visser et al. and Mehats et al. observed a

prolonged median survival in rat pups treated with rolipram only after birth (de Visser et al;

2008; Mehats C et al; 2008). It seems clear that rolipram is implicated in the maternal and

neonatal deaths in these studies, but further studies are needed to understand the mechanism

involved in these severe adverse events.

PDE4 inhibitors appear as a promising new class of anti-inflammatory drugs that have

shown efficacy in the experimental model of chorioamnionitis-induced bronchopulmonary

dysplasia described here. However, the conflicting results and side effects observed in

different experimental models tempered the enthusiasm for this treatment. Further


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investigations are needed in several different models before conclusions can be made

concerning the benefit/risk ratio of this treatment.


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ACKNOWLEDGEMENTS

The authors thank Mr. Arnaud LEGRAND, from his very helpful advice and suggestions


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AUTHORSHIP CONTRIBUTION

Participated in research design: LH, JHJ, GP, CGL

Conducted experiments: LH, CJ, CM, CGL

Contributed analytic tools: LH, CM, JC, PHJ, CGL

Performed data analysis: TM, BB, JCR, LH, CJ, CGL

Wrote or contributed to the writing of the manuscript: LH, NJ, EL, CM, GP, CGL


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LEGENDS FOR FIGURES

Figure 1. Perinatal mortality from inoculation to birth of pups. Data are presented as

relative value according to the time of death. Intrauterine death was significantly increased in

groups C+R+ and R+ as compared with both Controls and C+; #: p<0.03 for intrauterine

demise compared to controls and group C+. ¶: p<0.05 for neonatal demise compared to

controls. Neonatal death : hached bar, intrauterine death : solid black bars and live pups: white

bars.

Figure 2. Microvascularization and elastogenesis at day 5.

A: Immunohistochemistry labeling for microvascularization. Monoclonal antibody against

human CD31 was used. Data are percentage of labeled cells out of total parenchymal cells.

Microvascularization was significantly decreased in groups C+ (solid black bars), C+R+

(pointed bars), and R+ (hatched bars) compared to controls (solid white bars). Data are

presented as mean ± SD. # : p<0.01 compared to controls.

B: Specific elastine fiber length assessed by morphometry (cm/100g). Fiber length was

significantly decreased in groups C+, C+R+ and R+ as compared with Controls. Data are

presented as mean ± SD. # : p<0.05 compared to Controls.

Figure 3. Cell Turnover at day 0. A. Relative value of cell apoptosis over total cells in

lungs. Apoptosis was significantly decreased in group R+ (hached bars) as compared to

controls (solid white bars) and group C+R+ (pointed bars). Group C+ is presented as solid

black bars, data are presented as mean ± SD. # : p<0.05 compared to controls and C+R+B

PCNA index in terminal alveolar epithelium. B. Alveolar PCNA index: ratio of PCNA-

positive cells to total epithelial cells, expressed as a percentage. Data are presented as mean ±

SD. # : p<0.05 compared to controls.


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Figure 4. PDE activity in whole lung homogenates of rabbit pups exposed to

chorioamnionitis with or without rolipram. No significant difference in PDE activity was

found between the groups at day 0.

Control group:solid white bars, groups C+ :solid black bars, group C+R+ : pointed bars, and

R+: hatched bars. Data are presented as mean ± SD.


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TABLES


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Table 1. Morphometric measurements at day 0

Controls (n = 6)

C+ (n = 6)

C+ R+ (n = 6)

R+ (n = 8)

Body weight (g) 56.68 ± 9.3 47.18 ± 4.5 32.18 ± 6.1 39.90 ± 2.7

Lung volume (cm3) 2.89 ± 0.19 2.61 ± 0.26 2.32 ± 0.68 2.36 ± 0.33

Specific lung volume (cm3/100g)

5.23 ± 0.98 5.56 ± 0.62 7.09 ± 1.95 5.92 ± 0.73

Alveolar airspace

Volume density 0.66 ± 0.03 0.70 ± 0.02 0.64 ± 0.03 0.67 ± 0.01

Absolute volume (cm3) 1.93 ± 0.11 1.66 ± 0.48 1.49 ± 0.46 1.59 ± 0.22

Specific volume (cm3/100g) 3.50 ± 0.63 3.66 ± 0.78 4.54 ± 1.26 3.99 ± 0.47

Alveolar surface

Area density (cm2/cm3) 284 ± 34 225 ± 27 230 ± 84 223 ± 31

Absolute area(cm2) 822 ± 130 542 ± 193 520 ± 182 528 ± 108

Specific area (cm2/100g) 1485 ± 337 1187 ± 336 1660 ± 838 1320 ± 233

Interstitium

Volume density 0.28 ± 0.01 0.25 ± 0.02 0.28 ± 0.05 0.28 ± 0.02



Airways

Volume density 0.02 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.02 ± 0.01



Blood vessels

Volume density 0.02 ± 0.01 0.01 ± 0.01 0.04 ± 0.02 0.01 ± 0.01



Data are expressed as mean ± SD, comparison results are expressed with the F1/22 value accompanied with the

corresponding p value.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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Body weight; Infection effect F (1,22) =12.2, p=0.0002; Rolipram effect F (1,22) =27.7, p<0.0001. Interaction P>0.05

Alveolar airspace (volume density); Rolipram effect F(1,22) = 11.7, p=0.002; Interaction F (1,22)=16.0, p=0.0005

Alveolar airspace (specific volume), Rolipram effect F(1,22) =4.4, p=0,04

Interstitium (specific volume); Rolipram effect F(1,22) = 8.4, p=0.008;

Blood vessels (volume density); Interaction only F(1,22) = 6.8, p=0.05

Blood vessels (absolute volume ); Interaction only F(1,22) = 8.6, p=0.007

Blood vessels (specific volume );Rolipram effect F(1,22) = 4,7, p=0,04; Interaction F(1,22) = 7,8, p=0,01

All other comparisons not statistically significant




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Table 2. Morphometric measurements at day 5

groups day 5

Controls (n = 10)

C+ (n = 8)

C+ R+ (n = 5)

R+ (n = 10)

Body weight (g) 112.9 ± 10.8 72.7 ± 15.8 102.20 ± 14.5 70.89 ± 20.5

Lung volume (cm3) 5.99 ± 0.41 3.10 ± 0.62 5.15 ± 0.71 3.85 ± 0.86

Specific lung volume (cm3/100g) 5.34 ± 0.65 5.09 ± 0.95 5.05 ± 0.69 5.63 ± 1.13

Alveolar airspace

Volume density 0.60 ± 0.05 0.69 ± 0.03 0.60 ± 0.05 0.66 ± 0.06



Alveolar surface

Area density (cm2/cm3) 253 ± 25 223 ± 34 205 ± 5 220 ± 32

Absolute area(cm2) 1380 ± 249 862 ± 107 1057 ± 158 857 ± 253

Specific area (cm2/100g) 1313 ± 207 1214 ± 191 1034 ± 134 1221 ± 218

Interstitium

Volume density 0.33 ± 0.04 0.26 ± 0.01 0.30 ± 0.02 0.28 ± 0.05



Airways

Volume density 0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.03 0.02 ± 0.01



Blood vessels Volume density 0.05 ± 0.05 0.02 ± 0.01 0.04 ± 0.01 0.02 ± 0.01



Data are expressed as mean ± SD, comparison results are expressed with the F(1,21) value accompanied with the

corresponding p value.




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Body weight; Interaction effect only F (1,21) =44,6, p= 0,000001

Lung volume; infection effect F(1,22) = 7,6, p= 0,01; interaction effect F(1,21)=52,3, p=0,000001

Alveolar airspace (volume density); Interaction effect only F (1,21)= 10,4, p=0,003

Alveolar airspace (absolute volume), Interaction effect only F(1,21) =18,8, p=0,0002

Alveolar surface (area density); Rolipram effect F(1,21)= 11,7, p=0,002

Alveolar surface (absolute area ); Rolipram effect F(1,21)= 9 ,6, p=0,005

Alveolar surface (specific area ); Rolipram effect F(1,21)= 8,0, p=0,009

Interstitium ( volume density); interaction effect only F(1,21)= 6,1, p=0,02

Airways (absolute volume); interaction effect only F(1,21)= 10,9, p=0,003

Blood vessels (absolute volume ); Interaction only F(1,21) =5,5, p=0,02

All other comparisons not statistically significant




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Table 3: Effects of antenatal infection, Rolipram or both on bronchoalveolar lavage cell count

from day 0 to day 5

BAL cell count data are expressed as mean ± SD

Day 0 Day 5

Controls C+ C+R+ R+ Controls C+ C+R+

BAL cell count (X 104/ml)

1,3±1,0 1,6±1,7 2,4±0,1 0,8±0,2 0,9±0,9 1,2±1,1 5,1±0,2

PMN, % 0,51±0,16 0,88±0,45 0,91±0,22 1,22±0,67 2,07±1,24 2,45±1,56 3,66±1,89


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