•
Lung Growth and Lung Function Alter a) Fetal Lamb Tracheal Occlusion and
Exogenous Surfactant at Birth in Congenital Diaphragmatic Hernia and b) Selective
Perfluorocarbon Distention in Healthy Newborn Piglets
Andreana Bütter
Department of Experimental Surgery
McGill University, Montreal
•
Submitted June, 2001
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment
of the requirements of the degree of Master of Science
© Andreana Bütter, 2001
•
1+1
Nationallibrary
of·Canada
BibbolhèQue nationale
duClnadi
Acquisitions et
services bibliographiques
_.rueW~
0ftaM ON K1A ClN4
0INdlI
The author bas anmted a DODBdusiw licence aBowÎDI the
Natioual LiI:ny ofCuada to
œproduco, 1oaD, distribute or sen
copi. oItbis tbesis ÎD microfonn,
pep« or electrorûc formats.
The author ret- 0WMrSbip of the
copyriabt in tbis thesïs. Neitber the
thesis . . . . . . .tiaI exIrads hm il
may be priDted or otbawise
reproduced without the 8UthÔr's
permission.
L'auteur a acconIé une licence DOn
exclusive pametbmt à la
Biblioth6que DltiODlle du CaDada de
reproduire, Jdter. distribua- ou
vendre des caP- de celte daàe sous
la forme de mic:rofidleIfiJ de
reproduction sur papier ou sur format
Sectronique.
L'auteur CODIeI'W la propri6t6 ..
droit d' auteur qui P'Otèae cette".
Ni la th6se Di des eJdJills substantiels
de ceJle.ci De doiwDt an impIimés
ou autremeoI repJoduits saas son
autorisation.
0-612-B0111-X
Canadl
•
TABLE OF CONTENTS
Page
Abstract
3
Abrégé
4
Acknowledgements
5
Dedication
6
Abbreviations
7
Introduction
9
Review of the Literature:
A. Normal Lung Development
11
B. Normal Diaphragm Development
12
C. Pathophysiology of CDH
12
D. Animal Models ofCDH
14
E. Prenatal Interventions to Treat CDH
•
1. Tracheal Occlusion
14
2. Glucocorticoids
16
F. Postnatal Interventions to Treat CDH
1. Exogenous Surfactant At Birth
17
2. Selective Pulmonary Distention as a Means of Accelerating
Lung Growth
19
G. What Remains to Be Done
21
Materials and Methods
23
Results
34
Conclusion
38
Summary
43
References
44
Tables
54
Figures
56
•
2
•
ABSTRACT:
This study sought to maximize prenatal and postnatal interventions in order to
accelerate lung growth and improve lung function in two animal models. Prenatal
interventions consisted of fetal tracheal occlusion (TO), antenatal glucocorticoids and
exogenous surfactant at birth (SURF) in an ovine model of congenital diaphragmatic
hemia (CDH). CDH, CDH+TO, CDH+SURF, CDH+TO+SURF and unoperated twin
control lambs were compared. Prenatal growth of both lungs was accelerated after fetal
TO. Prophylactic surfactant did not improve gas exchange or ventilation but did increase
lung compliance over 8 hours. The incidence of tension pneumothoraces was slightly
decreased after exogenous surfactant. Fetal TO yields the best results in terms of overall
postnatallung function, likely acting via surfactant independent mechanisms.
Postnatal intervention involved perfluorocarbon (PFC) liquid distention of the
right upper lobe in healthy newbom piglets. Postnatal lung growth, as measured
indirectly by rates of DNA synthesis, was not accelerated after PFC distention.
•
•
3
•
ABRÉGÉ
Cette étude utilise des thérapies pré- et post-natales dans le but d'accélerer la
croissance pulmonaire tout en améliorant la fonction pulmonaire dans 2 modèles
animaux. Dans un premier temps, les effets de diverses therapies pré-natales, dont
l'occlusion de la trachée foétale (OT) et l'administration anténatale de glucocorticoides et
de surfactant (SURF), a été étudiée dans un modèle ovin de hernie diaphragmatique
congénitale (HDC). Cinq groupes expérimentaux ont été comparés: (a) HDC, (b)
HDC+OT, (c) HDC+SURF, (d) HDC+TO+SURF et (e) un groupe contrôle. Seule l'OT,
et non le traitment avec du surfactant, a accéleré la croissance pulmonaire. Malgré
l'addition de surfactant, seulement la compliance pulmonaire, et non l'échange gazeux ni
la ventilation, s'est amélioré au cours d'une période de huit heures. L'incidence de
pneumothorax est un peu moins après le surfactant. L'OT produit les meilleurs résultats
en ce qui concerne la fonction pulmonaire postnatale. On croit que des mécanismes
indépendants du surfactant sont responsables de ces améliorations.
Dans un deuxième temps, nous avons utilisé une thérapie post-natale dans le but
d'accélerer la croissance pulmonaire. La distension du lobe supérieur droit (LSD) a été
accomplie avec le liquide perfluorocarbon (PFC). Le taux de synthèse d'ADN n'a pas
augmenté de façon significative après ce traitement.
4
•
ACKNOWLEDGEMENTS
l would like to thank the following individuals for their invaluable help over the last year:
Dr. Hélène Flageole and Dr. Jean-Martin Laberge, my supervisors, for their constant
encouragement, incredible support and infectious enthusiasm throughout the year. Their
ability to transform potentially frustrating research experiments into an extremely
enjoyable and rewarding experience is remarkable and highly commendable!
Dr. Bruno Piedboeuf and Stéphane Guay for their work on the molecular aspects of this
project
Dr. Lajos Kovacs and Dr. Daniel Faucher for attending the numerous lamb resuscitations
and providing expert advice on neonatal anaesthesia, ventilation and surfactant
administration
•
Steve, Anie, Diana and Cynthia, the animal care technicians at the MacIntyre Animal
Ressource Centre, for their help with the lamb experiments throughout the year
Dr. Ioana Bratu for developing the resuscitation protocol in the CDH lamb and generating
the data on three of the experimental groups
Dr. Aurore Côté and Brian Meehan for their expert advice and assistance with the piglet
model
Dr. Xinying He for paraffin embedding, cutting and H & E staining the lung tissue from
both animaIs
Dr. David Bjarneson from BLES Biochemicals Inc. for graciously donating the BLES
surfactant
•
The Division of General Surgery at the University ofWestem Ontario for allowing me to
pursue my research year at the Montreal Children's Hospital
5
DEDICATION
To Sean, whom l love very much and who never let the 401 come between us!
•
•
6
•
ABBREVIATIONS
AaDO z = alveolar-arterial oxygen gradient
ABG = arterial blood gas
BAL = bronchoalveolar lavage
CDH = congenital diaphragmatic hernia
CMV = conventional mechanical ventilation
CPM = counts per minute
DBP = diastolic blood pressure
ECMO or ECLS = extracorporeal membrane oxygenation or life support
ET = endotracheal
FiOz = fraction of inspired oxygen
•
HR = heart rate
lM = intramuscular
IV = intravenous
LPS = left posterior segment of left upper lobe
LIS ratio = lecithin-sphingomyelin ratio
LUL = left upper lobe
LW/BW ratio = lung weight-to-body weight ratio
MAP = mean arterial pressure
MTBD = mean terminal bronchiole density
MVI = modified ventilatory index
NaHC03
•
=
sodium bicarbonate
01 = oxygenation index
PaCOz = partial pressure of arterial carbon dioxide
7
•
Pa02 = partial pressure of arterial oxygen
Paw = airway pressure
PEEP
=
positive end-expiratory pressure
PFC = per:tluorocarbon liquid
PIP = peak inspiratory pressure
PPHN = persistent pulmonary hypertension
RPS = right posterior segment of right upper lobe
RR = respiratory rate
RUL = right upper lobe
Sa02 = arterial oxygen saturation
•
SBP = systolic blood pressure
SEM = standard error of the mean
SURF = surfactant
VEI = ventilatory efficiency index
TO = tracheal occlusion
•
8
•
INTRODUCTION:
Congenital diaphragmatic hernia, or CDH, remams a challenging neonatal
condition to treat succesfully. CDH occurs in 1 in 2000-4000 live births (1-2). It
manifests around the 10th week of gestation when the fetal diaphragm fails to develop
properly. Persistent communication between the fetal chest and abdominal cavity results,
allowing intestinal contents to remain in the chest for the duration of the pregnancy.
Growth of both lungs is then limited by lack of intrathoracic space. At birth, the CDH
fetus possesses small, immature lungs which have difficulty adapting to extra-uterine life.
Acute respiratory failure often develops with death occuring in 30-50% of these neonates
(3). Despite many advances in neonatal care, mortality rates have remained at these
e1evated levels.
Prenatal ultrasound can diagnose fetuses with CDH in up to 93% of cases (4) and
as early as the 16th week of gestation (5). If the mother e1ects to continue with the
pregnancy, postnatal treatment with or without prenatal intervention is possible. Standard
•
postnatal care involves the use of mechanical ventilation, either conventional (CMV) or
high frequency (HFV), with or without nitric oxide (NO) followed by delayed repair of
the diaphragmatic defect (6-12). Extracorporeal membrane oxygenation (ECMO) may
also be required and can increase survival in selected patients (13-17). However, ECMO
is limited in that it cannot increase lung growth and the morbidity and mortality
associated with ECMO remains significant (18). Liquid distention, using perfluorocarbon
(PFC) liquid, is hypothesized to accelerate postnatal lung growth via stretch induced
mechanisms. For CDH neonates, increased lung growth could translate into decreased
time spent on ECMO with subsequent decreases in associated morbidity and mortality.
Postnatal distention could be offered to those newboms with CDH who were never
diagnosed prenatally and consequently, could never be offered fetal surgery.
Prenatal intervention, in the form of fetal surgery, is only indicated for poor
prognosisCDH babies i.e. those diagnosed prior to 25 weeks gestation with liver
hemiation into their chest and a lung-head ratio less than 1 (19-20). Although in utero
repair of the diaphragmatic defect has been attempted (21-22), its poor success rates led
•
ta its abandonment in favor offetal tracheal occlusion (TO) (23). Fetal TO, using either a
clip (24) or a balloon (25), occludes the trachea which increases intracheal pressure and
9
lung liquid volume, accelerating prenatal lung growth and improving postnatal lung
•
function (26~28). However, TO further exacerbates the CDH neonate's existing surfactant
deficiency by decreasing the density of type II pulmonary cells, the producers of
surfactant (29). By decreasing alveolar surface tension, surfactant facilitates alveolar
opening upon initiation of the first breath, thus decreasing the overall work of breathing
(30).
The goal of this study was to maximize both prenatal and postnatal interventions
in order to acce1erate lung growth and improve lung function in two animal models.
Prenatal interventions consisted of endoscopie fetal tracheal occlusion (TO) with or
without exogenous surfactant administered prior to the first breath in surgically created
CDH lambs. Postnatal intervention consisted of selective perfluorocarbon (PFC) liquid
pulmonary distention in healthy newbom piglets. AlI of these interventions could be used
in humans: fetaI TO and surfactant replacement at birth would be applicable to 'poor
prognosis' CDH neonates diagnosed prenatally while PFC distention could be used in the
•
management of either 'poor' or 'good prognosis' CDH neonates on ECMO.
Our hypotheses were as follows:
Hypothesis 1: Fetal TO in surgically created CDH lambs would acce1erate prenatallung
growth while the addition of exogenous surfactant at birth would further improve
postnatallung function.
Hypothesis 2: Liquid PFC distention
lU
healthy neonatal piglets would accelerate
postnatallung growth.
•
10
•
REVIEW OF THE LITERATURE
A) NORMAL LUNG DEVELOPMENT
To understand how CDH compromises lung function, we must first understand
normal lung development in humans and in our 2 animal models. The five stages
involved in normallung development (human / lamb / pig) are as follows (31-38), with
term being > 37 weeks in humans, > 145 days in lambs and> 115 days in pigs:
1. The Embryonic Stage (0-6 weeks / 0-40 days / 0-36 days)
A single lung bud, branching ventrally from the foregut, divides into 2 bronchial buds
which become the right and left mainstem bronchi. By the end of this stage, the trachea
and major airways down to the segmental bronchi are developed.
2. The Pseudoglandular Stage (7-16 weeks / 40-80 days / 36-55 days)
Ongoing branching of the bronchi results in complete development of the conducting
•
auways.
3. The Canalicular Stage (17-24 weeks / 80-120 days / 55-95)
The vascular system develops and the distal airways become further differentiated.
Maximallung growth occurs between 112 and 124 days in the sheep and 85 to 95 days in
the pig.
4. The Terminal Sac Stage (24-36 weeks /120-145 days / 95-115 days)
Differentiation of the future respiratory units (or acini) occurs along with type II cell
development. The gas-exchanging surface area increases dramatically while the bloodgas barrier decreases. Type II cells begin producing surfactant which consists of
phospholipids (often broadly referred to as lecithin) e.g. phosphatidylcholines (PC) and
phosphatidylglycerol (PG). Sphingomyelin, another type of phospholipid, is not produced
by type II cells but is 10cated within cell membranes. An increase in the lecithinsphingomyelin (LIS) ratio is crucial during this phase since an abnormal ratio is
•
predictive of respiratory distress syndrome at birth. Consequently, neonates born prior to
11
•
24 weeks gestation are unable to survive due to immature alveoli and lack of pulmonary
surfactant.
5. The Alveolar Stage (36 weeks-3 years postnatally / 145 days- /115 days- )
The number and size of alveoli increases significantly. Of note, only 15% of alveoli are
present at birth in humans. Alveolar multiplication occurs rapidly in the first 3 years of
life with more graduaI increases observed until g years of age. Conversely, in lambs and
in piglets, most alveoli are aIready present at birth.
B) NORMAL DIAPHRAGM DEVELOPMENT
The diaphragm is a musculotendinous structure that separates the thorax from the
abdomen. Four structures are required for its development: (i) the septum transversum,
(ii) the pleuroperitoneal membranes, (iii) the dorsal mesentery of the esophagus and (iv)
the lateral body walls (l, 39). Incomplete formation of the pleuroperitoneal membrane
and/or lack of fusion to the other three structures create a posterolateral diaphragmatic
defect. Although either side can be affected, most defects occur on the left side,
presumably due to earlier closure of the right pleuroperitoneal membrane (39). The
diaphragmatic defect is evident by the gth week in humans (40) and the 2g th day in both
lambs and pigs (35, 37). Abdominal viscera can then hemiate into the chest during
gestation and impair lung growth.
C) PATHOPHYSIOLOGY OF CDH
The high mortality associated with CDH is related to
its
complex
pathophysiology. Pulmonary hypoplasia, pulmonary hypertension, decreased pulmonary
compliance and surfactant deficiency aIl contribute to hypoxemia, hypercarbia and
acidosis (2). These factors stimulate pulmonary artery vasoconstriction, creating a vicious
cycle with worsening pulmonary hypertension, persistent fetal circulation with right to
1eft shunting and further deterioration of blood gases and acidosis (Figure 1). Therapeutic
interventions must target 1 or more areas in this cycle if outcome is to be improved.
Pulmonary hypoplasia is defined as a lung weight to body weight (LW/BW) ratio
less than 1.2% (41). The degree of pulmonary hypoplasia plays a key role in determining
12
•
the severity of respiratory compromise, and in fact, is the most important predictor of
survival in CDH (42-43). Although the ipsilateral lung is more severely affected, the
contralateral lung also demonstrates varying degrees of hypoplasia (44). CDH occurs
when abdominal viscera retum to the abdomen around the 10th week of gestation, with
sorne viscera hemiating into the chest and compressing the developing lungs. Airway
branching becomes arrested in the pseudoglandular stage in humans. Although the
surgically created CDH in lambs is performed in the canalicular stage, the end result is
similar to humans with a decreased LW/BW ratio, a decreased number of airway
branches, a decreased number ofalveoli and a thickened blood-gas barrier (41,45).
Another consequence of the compressive effect of the intestines on the developing
lungs is the incomplete development of the pulmonary vasculature (l, 46). Similar to the
bronchi, the pulmonary arteries develop fewer branches in both lungs (44). In addition,
the muscularization of the pulmonary arteries is increased. The end result is an elevated
pulmonary vascular resistance with right-to-Ieft shunting via a patent foramen ovale and a
•
patent ductus arteriosus (Figure 1) (44). The CDH baby cannot make the transition to
normal, neonatal circulation. Persistent pulmonary hypertension (PPHN) continues and
gas exchange is further worsened.
In addition, both animal and human CDH babies demonstrate altered lung
mechanics and amniotic fluid abnormalities similar to premature, surfactant-deficient
newboms. Pulmonary compliance is decreased in both lamb and human CDH neonates.
Amniotic
fluid analysis
(ÀF)
reveals immature
LIS ratios
and absent AF
phosphatidylglycerol (PG) in CDH neonates (47-49). A striking example of this was
shown in a case report by Hirthler et al (47). A set of diamniotic twins had an
amniocentesis performed at 38 weeks gestation to determine fetallung maturity. Twin A
(control) had a mature LIS ratio and positive PG whereas twin B (CDH baby) had an
immature LIS ratio and negative PG. At birth, twin B was diagnosed with CDH.
However, in CDH lambs, amniotic fluid LIS ratios and PG levels were similar to control
lambs. On the other hand, bronchoalveolar lavage (BAL) fluid from CDH lambs did
demonstrate decreased levels of surfactant proteins (50).
•
13
D) ANIMAL MODELS OF CDH
•
Various animal species with either medical or surgical creation of CDH have been
used to examine the effects of CDH on lung structure and function (51). The teratogenic
rat model, where nitrofen is given to induce CDH, is used to examine the embryological
and molecular aspects of lung structure. However, nitrofen damages many organs
including the lungs. Thus, lung development is adversely affected by both the nitrofen
and the CDH (51). Surgically created CDH in lambs enables investigators to study the
efficacy of therapeutic interventions on lung function and structure. The disadvantage of
this approach is that the CDH is created at a 1ater gestational age in the lamb (ie. during
the canalicular stage) than occurs in humans. However, as previously mentioned, the
pathophysiologic changes associated with CDH in this ovine model remain significant.
Histologic and physiologic studies have revealed that the surgically created CDH lamb
model closely mimics human CDH neonates (52-54).
The ideal animal model would possess a naturally-occurring diaphragmatic
•
hernia. One such model has been described in a herd of piglets originally being bred for
anorectal anomalies (55). However, the diaphragmatic defect is not consistent in its
location and a large number of animaIs are required to obtain a sufficient number of CDH
pig1ets.
E) PRENATAL INTERVENTIONS TO TREAT CDH
1. TRACHEAL OCCLUSION
Laryngeal atresia, a rare congenital malformation, was incidentally noted to
produce large, hyperplastic and structurally mature lungs at birth (56). This observation
led to the idea that experimental occlusion of the trachea in CDH fetuses may reverse its
associated pulmonary hypoplasia. Tracheal occlusion studies in fetal lambs have
demonstrated true pulmonary hyperplasia (25-26, 57-61). Both Alcorn (57) and Flageole
(25) have shown doubling of the LW/BW ratio in healthy lambs after TO. Wilson (59)
demonstrated a 4 fold increase in lung volume to body weight ratio and total alveolar
surface area. Histologically, these hyperplastic lungs appear structurally mature (25, 59).
•
Nardo (26), in an ovine model of pulmonary hypoplasia induced by chronic lung liquid
drainage, has shown that reversaI of hypoplasia occurs after 6 days of TO. Increased
14
DNA synthesis rates, an indicator of lung cell number, are already present by day 2 of
•
Ta, begin to decrease on day 4 and have returned to controllevels by day 10 (60).
Applying this technique to a fetal CDH lamb model, lung growth is again
demonstrated although to a lesser degree than in normal lungs. Hedrick demonstrated
doubling of the dry lung weight in CDH + Ta lambs compared with CDH lambs (23).
Benachi (27) showed that endoscopie Ta in CDH lambs significantly increased their
LW/BW compared with both CDH only and controllambs.
In human CDH babies, Ta also results in increased prenatal lung growth and
reversaI ofpulmonary hypoplasia at birth (61).
How does fetal tracheal occlusion cause this accelerated lung growth? Several
theories have been formulated although the exact mechanism of action remains unknown.
Increased intratracheal pressure has been measured during fetal Ta (28, 62). Nardo (60)
measured both tracheal pressure and lung liquid volume during Ta. Although tracheal
pressures increased during the 1st day of Ta, they subsequently plateaued for the
•
remaining 9 days but remained above normal levels. In contrast, lung liquid volume
continued to increase until day 7 and then stabilized until day 10. The authors concluded
that an increase in lung liquid volume may be more important that increased tracheal
pressures to accelerate lung growth prenatally. However, Kitano (62) demonstrated that
by maintaining tracheal pressures above the usual 4-5 mm Hg observed during Ta,
prenatallung growth could be further increased compared with Ta alone.
Fetallung growth is dependent on lung liquid volume, fetal breathing movements,
sufficient intrathoracic space and amniotic volume (63). Lung liquid volume is critical for
adequate lung growth. Fetal respiratory epithelial cells produce lung liquid at varying
rates throughout gestation (63). However, at Ils days gestation in the sheep, there is a
dramatic increase in both tracheal pressure and lung liquid volume (63). Thus, Ta
prevents egress of lung liquid during maximal liquid production. This increased volume
leads to increased pressure which stimulates alveolar and epithelial cell proliferation. An
increase in epithelial cell density translates into a further increase in lung liquid volume
with the end result being accelerated lung growth. This positive cycle continues as long
•
as the Ta stimulus is maintained. The degree of lung growth is dependent on the length
15
of the occlusion: lambs with TO for only 1 or 2 weeks have less lung growth than those
•
with TO for 3 weeks (29, 64).
Other groups have advocated that growth factors in the lung liquid are responsible
for this increase in pulmonary growth. Papadakis (65) aspirated tracheal fluid from 2 sets
of healthy fetallambs with TO and either replaced it with saline (group 1) or reinfused it
back into their trachea (group 2). Group 1 did not develop lung hyperplasia. The authors
concluded that TO appears to be mediated by growth factors within the tracheal fluid as
opposed to increased intratracheal pressure and/or volume. Many growth factors,
including transforming growth factor pl and p2 (TGF-pl and TGF-P2), insulin-like
growth factors (IGF-I and IGF-II), epidermal growth factor (EGF) and platelet-derived
growth factor (PDGF), are being studied as potential mediators of fetallung growth (6667).
Besides accelerating prenatal lung growth, TO also prevents excess pulmonary
artery muscularization in CDH rats (68) and lambs (69). Our group has recently shown
•
that TO thins the medial area of small pulmonary arteries in CDH lambs (69). These
structural changes help to decrease pulmonary hypertension which leads to improved gas
exchange, ventilation and compliance (69).
Unfortunately, TO accelerates lung growth at the expense of type II cells, the
producers of surfactant (29). It is postulated that the stretch induced by TO stimulates
type II cells to convert into type 1cells (70). Tracheal release (TR) has been advocated as
a means of preventing this decrease. Prior work in our laboratory has demonstrated
recovery of type II cell density with TR performed 2 days prior to delivery in normal
lambs (71) and 1 week prior to delivery in surgically created CDH lambs (72). However,
surfactant levels remained low, suggesting that exogenous surfactant at birth may be
beneficiaL
2. GLUCOCORTICOIDS
Prenatal glucocorticoids improve postnatallung function in normal animal models
and humans and in CDH lambs. In normal sheep, a single fetal or maternaI injection of
•
betamethasone improved PaOl, increased lung volumes and improved pulmonary
compliance (73-75). Although glucocorticoids have been shown to increase lung
16
surfactant levels, the improved pulmonary compliance observed postnatally appears to be
•
due, in part, to surfactant-independent mechanisms (76-77). Fiascone demonstrated that
preterm rabbits given prenatal steroids and exogenous surfactant had increased lung
compliance compared with rabbits given only steroids or surfactant. However, alveolar
surfactant levels were similar for steroid + surfactant rabbits and surfactant only rabbits
(76). Prenatal betamethasone injections in rhesus monkeys increased lung volumes and
alveolar distensibility but did not affect amniotic or pulmonary surfactant levels (77).
Antenatal glucocorticoids, in combination with tracheal occlusion and release, increased
type II cell density to controllevels in healthy lambs (71) but not in CDH lambs (72).
Thus, it appears that structural remodeling, as well as surfactant levels, improves
postnatal lung function by altering collagen:elastin ratios (77) and decreasing alveolar
wall thickness (78) after prenatal glucocorticoid treatment. These structural changes
increase alveolar distensibility, which leads to greater lung volumes and improved
compliance. Similar changes are observed in surgically created CDH lambs and nitrofen-
•
induced CDH rats: alveolar wall thinning and increased alveolar volume are
demonstrated after antenatal steroid treatment (79-80).
In both normal and CDH human lungs, glucocorticoids also accelerate pulmonary
maturity, improve compliance, decrease vascular leakage, improve lung liquid clearance,
increase endogenous surfactant and enhance the neonate's response to exogenous
surfactant administration (81).
Finally, the route, dose, timing and duration of steroid administration are also
important. For example, lower doses of steroids (i.e. 0.2 mg/kg instead of 0.5 mg/kg)
given too close to delivery (i.e. 8 hours before birth instead of 15 hours) do not improve
postnatallung function in preterm lambs (73-74).
F) POSTNATAL INTERVENTIONS TO TREAT CDH
1. SURFACTANTREPLACEMENTTHERAPY
Both the surgically created CDH lamb and the nitrofen-induced CDH rat are
surfactant deficient, with low surfactant phospholipid levels in BAL fluid and lung tissue
•
compared with control animaIs (50, 82). In contrast, the presence of a surfactant
deficiency in CDH human neonates is controversial. Physiologically, CDH newboms
17
behave in a similar fashion to premature, surfactant deficient babies with poor pulmonary
•
compliance and hyaline membrane formation (30). Case reports have documented
decreased LIS ratios in the amniotic fluid of CDH pregnancies (46-48). However, BAL
fluid in CDH newboms demonstrated similar surfactant phospholipid concentrations
compared with age-matched controls (83). Thus, the surfactant deficiency associated with
CDH may not be primary in nature but rather, occur secondarily after the onset of
respiratory failure (83). Nevertheless, the overwhelming success of surfactant
replacement therapy in premature newboms with respiratory distress syndrome (RDS)
has prompted its application to numerous pulmonary conditions including CDH (30).
Exogenous surfactant can be administered either prophylactically i.e. before the
first breath, or as rescue therapy i.e. after the neonate develops symptoms of respiratory
distress (30). In premature newboms less than 30 weeks gestation, prophylactic surfactant
administration resulted in increased survival compared with surfactant given as rescue
therapy (84).
•
Prophylactic surfactant administration significantly improves gas exchange and
lung mechanics in CDH lambs and humans (54, 85-87). In CDH lambs, Wilcox (54)
demonstrated improved Pa02, decreased PaC02, increased pH and increased compliance
in CDH + surfactant lambs compared with CDH lambs at 30 minutes of life. However,
the absence of a control group makes the significance of these changes difficult to assess.
Additional experiments by the same group examined clinical outcome after 4 hours of
resuscitation between 3 groups: (i) CDH lambs with antenatal repair of their
diaphragmatic hemia, (ii) CDH + TO lambs and (iii) CDH + TO + surfactant
administered prior to the 1st breath. Both PaÜ2 and pulmonary blood flow were improved
during the 4 th (and last) hour of resuscitation in the surfactant group only (88). The
authors concluded that fetal Tü may not produce a physiologically normal lung and that
surfactant and/or prenatal steroids may be sufficient treatment for improved postnatal
lung function. But again, the lack of both normal controls and CDH control lambs
undermines these results.
As for CDH humans, Glick (87) treated 3 high risk prenataHy diagnosed CDH
•
newboms with prophylactic surfactant. Despite many adverse prognostic factors and an
expected survival rate of less than 20%, aH 3 babies survived. The authors concluded that
18
surfactant prophylaxis appears promising in CDH neonates and a randomized, controlled
trial should be performed.
Altemative1y, for those infants whose CDH is only diagnosed postnatally when
they develop respiratory distress, exogenous surfactant can be administered as rescue
therapy. In CDH lambs, surfactant rescue had no effect on PaC02, Pa02 or pH (89). An
increase in pulmonary blood flow was observed but this was not significant. The authors
concluded that if exogenous surfactant is being considered in the CDH neonate, it should
be given as soon as possible, preferably prior to the first breath (89). In human CDH
neonates, Lotze administered 4 doses of bovine surfactant as rescue therapy to 9 CDH
newboms on ECMO. No changes in lung compliance, duration of oxygen therapy or
length ofhospital stay were observed when compared with CDH neonates (90). However,
Bos (91) administered surfactant as rescue therapy once RDS symptoms had developed.
In 2 of the 4 patients, AaD0 2 dropped dramatically along with Fi02 and PIP. However,
repeated doses had only a transient effect on oxygenation. These 2 newboms survived
•
while the remaining 2 did not demonstrate any improvement in their oxygenation and
succumbed to their disease.
In general, exogenous surfactant appears to be well tolerated (30). Although
exogenous surfactant has been argued as predisposing patients to an increased incidence
of pneumothoraces, a recent meta-analysis of randomized, controlled trials demonstrated
a decreased incidence of pneumothorax in premature infants with RDS receiving
prophylactic surfactant (92).
2.
SELECTIVE
PULMONARY
DISTENTION
AS
A
MEANS
OF
ACCELERATING LUNG GROWTH
Although many types of postnatal treatment currently exist for CDH neonates
with respiratory failure including CMV (1), HFV (2), ECMO (14) and exogenous
surfactant (30), all are limited by their inability to acce1erate lung growth. These
therapeutic modalities can only support the CDH neonate until their pulmonary
barotrauma resolves or their pulmonary hypertension improves. In cases of severe lung
•
hypoplasia, these modalities remain ineffective (42-43). If lung growth could be
accelerated postnatally, these affected infants might increase their totallung mass beyond
19
that required for survival (37). It has been theorized that pulmonary liquid distention,
•
acting via similar stretch-induced mechanisms as fetal TO, could accelerate lung growth
postnatally.
The search for the ideal substance involved experimentation with many different
types of liquids inc1uding saline (93) and natural oils (94). However, each possessed
major drawbacks: saline had low oxygen solubility and washed out surfactant (95) while
natural oils were too viscous and toxic (94). The search for a more appropriate fluid led
to the discovery of perfluorocarbon (PFC) liquids (93). Although sorne fluorocarbons
were produced as early as World War II, their use as a respiratory medium was not
discovered until the 1960's (96). PFC possesses many attractive characteristics: it is c1ear,
colorless, odorless, inert and non-toxic in addition to being highly soluble for oxygen
(02) and carbon dioxide (C0 2) (97). PFC's high density enables it to reach atelectactic
areas of the lung and recruit additional alveoli. Like surfactant, PFC possesses a low
surface tension which improves lung function and gas exchange. Pulmonary blood flow
•
is also redistributed towards the more ventilated, less dependent areas of the lung, which
decreases ventilation-perfusion mismatch (97).
PFC, when used as a respiratory medium during liquid ventilation in CDH
animals/neonates, has resulted in sorne improvements in gas exchange and compliance
but does not accelerate lung growth or increase survival (98-100). In contrast, selective
PFC distention, although only performed in sheep to date, appears promising with respect
to increasing postnatallung growth (97, 101). Nobuhara (97) used continuous PFC liquid
distention in the right upper lobe (RUL) ofneonatal and adult sheep lungs for 21 days. A
significant acceleration in neonatal lung growth was demonstrated by an increased RUL
volume to body weight ratio, an increased total alveolar number and an increased total
alveolar surface area compared to controls. Similar experiments in aduIt sheep did not
reveal any evidence of accelerated lung growth (97).
Nobuhara (lOI) then examined the long-term effects of liquid pulmonary
distention in sheep. After 7 days of PFC distention in the RUL, the lamb's right bronchus
was reconnected and spontaneous respiration was resumed. The animaIs were sacrificed
•
at 3 to 6 months. Lung growth was not increased. The authors conc1uded that 1 week of
postnatal liquid distention was insufficient to accelerate lung growth and this technique
20
was therefore not clinically relevant. However, their 'neonatal' lambs were 4 weeks old
at the onset of their experiments. By this age, the thoracic cage is already fairly rigid
which limits the space available for lung growth. In addition, they failed to consider the
possibility that accelerated growth may have occurred initially but then plateaued once
the PFC stimulus was removed. As previously mentioned, fetal TO increases DNA
synthesis rates by day 2 to 280% but decreases to controllevels by day 10 (26).
Selective pulmonary distention using PFC liquid offers a pro-active means of
accelerating lung growth postnatally in neonates. This technique would be especially
helpful in CDH babies whose severe lung hypoplasia requires ECMO. Although lung
growth does occur in these patients, it is usually too slow to enable long term survival.
Continuous PFC distention could be used to accelerate lung growth while the CDH
neonate receives ECMO to ensure adequate gas exchange. Subsequent increases in lung
growth could translate into more rapid weaning from ECMO. Over the long term, this
technique could lead to decreased morbidity and mortality rates and offer an additional
•
effective treatment to patients and their families.
G) WHAT REMAINS TO BE nONE
Since CDH may not always be diagnosed prenatally, we wished to maximize both
prenatal and postnatal therapeutic interventions in 2 animal models. To date, no study has
examined the effects of prenatal tracheal occlusion in combination with antenatal
glucocorticoids and prophylactic surfactant at birth in surgically created CDH lambs
compared with controls over an 8 hour resuscitation period. Each of these interventions
targets one of the aspects of CDH's pathophysiology: TO accelerates prenatal lung
growth and reverses pulmonary hypoplasia while glucocorticoids and exogenous
surfactant increase surfactant levels, which improves lung compliance and function.
However, sorne CDH neonates are only diagnosed postnatally and thus cannot benefit
from prenatal TO, antenatal glucocorticoids or prophylactic. surfactant therapy.
Consequently, postnatal interventions are also important. To our knowledge, no one has
examined the short-term effects of PFC distention on lung growth in healthy neonatal
•
piglets. Ifbeneficial, PFC distention could be applied to our surgically created CDH lamb
model and eventually, to human CDH neonates. Although the use of the piglet introduces
21
interspecies variation, both piglets and lambs are feh to be similar in terms of pulmonary
•
anatomy and function (33, 38). In fact, both possess a right tracheal bronchus to their
RUL, which facilitates selective pulmonary distention. In addition, piglets are more
readily available and at a significantly reduced cost compared with lambs.
•
•
22
MATERIAL8 AND METHOD8
•
EXPERlMENT #1: FETAL TRACHEAL OCCLUSION & EXOGENOUS
SURFACTANT AT BIRTH INA SURGICALLY CREATED CDH LAMB MODEL
A) ANIMAL MODEL
1. Creation ofCDH
Ethics approval for aIl animal experiments was obtained from the McGill University
Animal Care Committee. Using previously described techniques (103), a left sided CDH
was created in the fetal lamb at 80 days gestation. The time-dated pregnant mixed breed
ewe was fasted 24 hours before surgery. The ewe was anaesthetized with IV thiopental
and anaesthesia was maintained with an oxygenated-halothane mixture and mechanical
ventilation. The ewe's abdomen was shaved and cleaned with proviodine soap and
alcohol followed by a proviodine prep solution. The abdomen was then draped and strict
asepsis was enforced. Using a midline laparotomy, the uterus was exposed and delivered
through the incision. The position and number of fetuses was determined. The fetal parts
•
were palpated and the fetal left hemithorax was identified using the head, spine, shoulder
blade and costal margin as landmarks. Two 4-0 silk stay sutures were placed through the
uterus and into the fetus' chest wall to maintain fetal position. A small hysterotomy was
made over the lower left chest. A left fetal thoracotomy exposed the left lung and
diaphragm. A cotton tip applicator was used to gently retract the fetallung and identify
the central white fibers of the diaphragm. A 23G needle tip was used to puncture the
diaphragm while fine scissors and small mosquito forceps were used to enlarge the defect
to a diameter of 1-1.Scm. At least 2 of the 3 stomachs were carefully pulled up into the
left chest. The fetal chest was closed with an interrupted 4-0 silk suture to re-approximate
the fetal ribs and a running 4-0 silk suture to close the fetal muscle and skin layers. A 4-0
silk stitch was placed in the fetal chin for identification during future procedures.
Warmed normal saline along with Cefazolin SOOmg, Gentamycin 1S0mg and Liquamycin
200mg were infused into the amniotic cavity prior to uterine closure. The ewe's
abdominal fascia was re-approximated with interrupted #1 Pro1ene sutures. Running 3-0
Vicryl and running subcuticular 3-0 Vicryl were used to close the subcutaneous tissue
•
and skin, respectively. The incision was sprayed with Opsite. Each ewe was given
liquamycin 400 mg lM daily for the first 3 post-operative days.
23
2. Tracheal occlusion (TO)
•
Fetal tracheoscopy (2.7mm Semi Flexible Mini-Endoscope, Karl Storz, Germany)
was used in combination with a detachable balloon system (GVB12 Latex Goldvalve
Balloon, maximum diameter 14mm, length 22.5mm, volume 2.5ml; CCOXLS co-axial
catheters) to achieve fetal tracheal occlusion (25, 104-106). The time-dated pregnant ewe
at 108 days gestation was anaesthetized and prepared for laparotomy as described above.
The gravid uterus was delivered through the midline abdominal incision and the fetal
head and mouth were palpated through the uterine wall. A surgical assistant extended the
fetal neck in order to facilitate endoscopie entry. A small hysterotomy was made directly
over the fetal snout and the endoscope was advanced into the fetal mouth. Amniotic fluid
loss was prevented by using a 2-0 Vicryl purse string suture to create a seal between the
endoscope and the uterine wall. Infusion of warmed saline through the first channel of the
endoscope improved visibility and facilitated passage of the endoscope through the vocal
cords and into the trachea. Using the second channel of the endoscope, the balloon was
•
advanced into the trachea, placed approximately 2 cm below the vocal cords,filled with
1.5 ml of methylene blue dyed saline and detached from its catheter. Prior to removing
the endoscope, the detached balloon was visualized to ensure that its valve was closed
and there was no leakage of blue saline. Cefazolin, Gentamycin and Liquamycin were
placed in the uterus prior to closure of the purse-string hysterotomy in 2 layers. The
remainder of the abdominal closure was completed as previously described.
3. Delivery and resuscitation protocol
At 129 days gestation, aIl ewes received 250mg medroxyprogesterone lM to prevent
preterm labour (107). Progesterone is not known to affect lung development (108). At
135 days gestation, aIl ewes received 0.5mg/kg betamethasone lM since the lambs are
delivered prematurely (73-74, 109).
At 136 days gestation, the fetallamb was delivered by cesarean section (98). The ewe
was anaesthetized as described above. A midline 1aparotomy was performed and the
gravid uterus delivered through the incision. A hysterotomy large enough to deliver only
•
the fetal head and neck was made. A sterile latex glove filled with warm saline was
immediately placed over the 1amb's snout to prevent spontaneous breathing. With the
24
lamb still under placental circulation, a transverse incision in the fetal neck
•
approximately halfway between the thyroid cartilage and suprasternal notch enabled a
limited dissection of the trachea, rightjugular vein and right carotid artery. In CDH lambs
with TO, the trachea was palpated to determine the position of the endotracheal balloon.
The trachea was encirc1ed with 2 pieces of umbilical tape distal to the balloon and the
proximal piece was tightened to prevent balloon migration. A tracheostomy was
performed between the 2 pieces of umbilical tape and the pulmonary fluid was
immediately suctioned into a suction trap. Based on our previous experiments with
lambs, we presumed an average birth weight of 3 kg to calculate the doses of medications
that needed to be given before the animal could be weighed. The first dose of BLES
surfactant (5ml/kg or 15 ml) was then injected through an 8Fr feeding tube positioned
just above the carina. A 3.5 - 4 mm (LD.) uncuffed and occ1uded endotracheal (ET) tube
was positioned to the 2 cm mark and then secured with umbilical tape. We then
proceeded to cannulation of the vessels. The right jugular vein was distally ligated with
•
2-0 silk, a 5.5Fr triple lumen catheter was inserted down to the lOcm mark and firmly
secured with 2-0 silk. Each lumen was flushed with heparinized saline
CI U/ml). The
carotid artery was then distally ligated with 2-0 silk, an 180 Jelco catheter was inserted
and secured in place with 2-0 silk. A baseline arterial blood gas was immediately drawn.
The arterialline was then flushed with heparinized saline CI U/ml). Pancuroniurn bromide
0.3 mg IV (0.1mg/kg), ketamine 18 mg IV (6mg/kg) and sodium bicarbonate (NaHC0 3)
6 mmol IV (2 mmol/kg) were given through the jugular venous line for paralysis,
analgesia and correction of acidosis respectively. Enlargement of the hysterotomy
allowed the entire fetus to be delivered. After c1amping the umbilical cord and
unc1amping the ET tube, the lamb was immediately bagged with oxygen, weighed and
placed under a radiant overhead warmer.
Prior to euthanizing the mother, 60 ml of placental blood was collected in a preheparinized syringe and stored on ice for possible lamb transfusion (although tbis was
never required in this set of experiments). MaternaI euthanasia was achieved with an
overdose of IV pentothal.
•
Simultaneously, the lamb was started on mechanical ventilation (Sechrist Infant
Ventilator Model IV-1 OOB) along with infusions of pancuronium
25
CI mg/kg/h), ketamine
•
(2 mg/kg/br) and NaHC03 (0.5 mmol/kglhr) via the jugular venous line. Both the central
venous and arteriallines were continuously flushed with 0.5 mllbr of heparinized saline
(1 U/ml) to maintain patency. The ventilator was initially set as follows: peak inspiratory
pressure (PIP) 15 H20, peak end-expiratory pressure (PEEP) 5 cm H20, Fi02 1.0,
respiratory rate (RR) 120 breaths per minute, inspiratory time 0.25 sec and inspiratory
time to expiratory time ratio 1: 1. In order to maximize gas exchange and minimize
airway pressure, ventilatory settings were adjusted during the experiment if PaC02 < 40
mm Hg or > 65 mm Hg; ifPa02 < 40 mm Hg or> 100 mm Hg; and ifpH < 7.4 or> 7.5.
Boluses of NaHC03 2 mmol/kg IV were given to increase the pH by 0.1 unit when pH
was < 7.4.
Heart rate (HR), oxygen saturation (Sa02, postductal), central venous pressure,
systolic (SBP), diastolic (DBP) and mean arterial (MAP) blood pressures and rectal
temperature were continuously monitored. The lamb's temperature was maintained
between 38-39°C using an overhead warmer and a heating pad. A percutaneous
•
cystostomy, consisting of suprapubic insertion of an 180 Jelco catheter, was used to
monitor urine output., Arterial blood samples were taken as follows: an initial sample was
taken prior to clamping the umbilical cord followed by one taken every 15 minutes for
the first hour of life, then every 30 minutes for the second hour of life, and finally every
hour until completion of the 8 hour resuscitation. A portable clinical analyzer and E07+
cartridges (i-STAT, Sensor Deviees Inc., Waukesha, WI, USA) were used to determine
blood glucose, electrolytes (sodium, potassium, calcium), hemoglobin, hematocrit and
pre-ductal arterial blood gas values (Pa02, PaC02, pH). Potassium levels less than 3
mmollL were corrected with a slow intravenous bolus of 1 mEq of KCl. Ionized calcium
levels less than 0.9 mmoliL were treated with 1 ml/kg IV bolus of 10% calcium
gluconate. Blood glucose was also checked regularly for either hypoglycemia (glucose <
2.0 mmollL) or hyperglycemia (glucose> 20 mmol/L) although this never occurred. One
dose of antibiotics was given in the first hour of life tbrough the jugular venous line
(Ampicillin 50 mglkg IV, Gentamycin 2.5 mglkg IV).
Respiratory function was initially assessed at 1.5 hours of life and then after every
•
blood gas sample for a minimum duration of 1 minute. Tracheal pressure, flow and
volume were measured using a pneumotachometer and pulmonary function machine
26
(Raytech Instruments Inc., Vancouver, BC, Canada). Respiratory compliance was
calculated as the change in volume over the change in pressure during no flow states in
the respiratory cycle.
A tension pneumothorax was suspected if the MAP was < 40 mm Hg or the blood
pressure decreased by > 50%. Rapid visual inspection of the thorax for hyperinflation and
lung auscultation for decreased breath sounds usually revealed the affected lung.
Insertion of a 10-12Fr chest tube to straight drainage into the fifth intercostal space in the
mid-axillary line on the afflicted side would relieve the pneumothorax. Howevet, if the
lamb remained hemodynamically unstable, a contralateral chest tube was inserted. If the
lamb' s cardiopulmonary status was still not improved, a tension pneumomediastinum
was suspected and relieved with a subxiphoid incision through the pleura of the
mediastina1 lobe (part of the normal anatomy of lambs) and placement of a third chest
tube. According to our protocol, an epinephrine bolus of 0.1 mg/kg was to be given if
MAP < 20 mm Hg but this was never required.
•
A second dose of BLES surfactant (5 m1lkg) was given at four hours of life. The
lamb was disconnected from the ventilator and manually ventilated for 30 seconds. A
pre-measured 8Fr feeding tube was inserted down the ET tube. The surfactant was given
in 3 aliquots with the lamb in the following positions: on the left side, on the right side
and supine. These changes in position enabled a uniform distribution of BLES to aIl
pulmonary lobes. After each aliquot, the lamb was manually ventilated for 1 minute
while inspecting the chest to ensure good chest expansion with every breath. Upon
completion of the second dose, the lamb's ET tube was reconnected to the ventilator.
4. Terrnination ofresuscitation:
AlI 10 animaIs in this set of experiments survived the entire 8 hour resuscitation.
However, previous work in our lab with CDH only lambs had 2 deaths prior to
completion of the 8 hour time frame. These deaths were preceded by:
a) HR < 80 for 30 minutes
b) MAP < 20 mm Hg despite blood transfusions, fluid boluses, epinephrine bolus and
chest tubes
c) pH < 6.8 on 3 consecutive arterial blood gases done one hour apart
27
5. Neonatallamb euthanasia and autopsy:
•
At the end of the experiment, while still under general anaesthesia, the lamb was
euthanized with an overdose of IV pentothal. A midline stemotomy exposed the thoracic
contents. The presence, size and position of the diaphragmatic hemia was recorded along
with the type and amount of viscera hemiating through the defect. The position of the
endotracheal balloon was also noted when applicable. The trachea was dissected and the
heart and lungs were removed en bloc. Totallung weight, followed by right and left lung
weight, were measured after cutting the left main bronchus. Total, right and left LWIBW
were ca1culated. The dry-to-wet lung weight ratio (111) was determined from samples
taken from the right middle lobe and lingula. These lung samples were weighed
immediately after resection, left to air dry for one week and then re-weighed. Mu1tiplying
the wet LW/BW by the dry-to-wet lung weight ratio yielded the dry LWIBW.
B) EXPERIMENTAL GROUPS AND OUTCOME MEASURES
•
Previous work in the lab using identical protocols produced non-operated controls
(n=4), CDH lambs (n=5) and CDH + TO lambs (n=5), aIl of whom never received
exogenous surfactant. These groups were compared with CDH + SURF lambs (n=4) and
CDH + TO + SURF lambs (n=6) using the following outcome measures:
1. Survival
2. Lung growth: LW/BWratio
3. Arterial blood gas trends: pH, PaC02, Pa02
4. Oxygenation and ventilatory parameters (115):
a) Alveolar-arterial oxygen gradient (AaDOz) = [((713xFiOz)-PaCOz)/O.8]-PaOz
b) Modified Ventilatory Index (MVI) = (RR x PIP x PaCOz)/1000
c) Ventilatory Efficiency Index (VEI) = 3800/((PIP-PEEP) x RR x PaCOz
d) Airway Pressure (Paw) = PIP - PEEP
e) Modified Oxygenation Index (01) = (Paw x Fi02 x 100)/Pa02
5. Lung compliance
•
28
C) STATISTICAL ANALYSIS
The statistical software package SPSS version 10.05 enabled comparison of the 5
groups using ANOVA with Bonferoni or Dunnett's post-hoc testing at each time point
and over time. Survival data was compared between groups using a chi-square test with
Yates' correction. The right and left lungs were compared using paired t-tests. Statistical
significance was reached when p:s; 0.05 .
•
•
29
•
EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC
LIQUID IN HEALTHY, NEONATAL PIGLETS
A) ANIMAL MODEL
1. Experimental design
Piglets, aged 5 - 8 days, were obtained from a farm in St. Lazare (weIl known
supplier for other MCH researchers) and were randomly divided into 4 experimental
groups: (a) non-operated controls (n=4), (b) operated controls (n=4), (c) PFC x 6 hrs
(n=lO) and (d) PFC x 12 hrs (n=6). Operated piglets were anaesthetized with 0.5-2.5%
halothane-oxygen-nitrous oxide mixture by mask, titrated to effect. They remained under
general anaesthesia for the duration of the experiment. A tracheotomy was performed
using a transverse incision midway between the thyroid cartilage and the suprastemal
notch. The cricoid cartilage was identified and the trachea incised between its third and
fourth rings. After placement of a 3 mm (LD.) ET tube secured with umbilical tape,
mechanical ventilation (CMV) was initiated. Ventilatory parameters were as follows:
•
frequency 40 breaths/min, pressures 16/5 cm H20, Fi02 0.4. Lateral extension of the neck
incision revealed that both the carotid and jugular veins were too small for cannulation.
Our protocol was then modified to include dissection of the right superficialaxillary vein
and the right superficial femoral artery. Both were distally ligated with 3-0 silk,
cannulated with 3Fr umbilical catheters and secured with 3-0 silk ties. The axillary vein
catheter was used to administer medications and infuse a maintenance electrolyte and
glucose solution (NaCl 0.2 + 5% dextrose @ 25ml/hr). The femoral arterialline was used
to obtain arterial blood gas samples. Its patency was ensured by continuous flushing with
0.5mllhr of 1U/ml heparin in normal saline. The neck incision was closed with
interrupted 3-0 silk sutures. Cloxacillin (50mg/kg IV) and Gentamycin (2.5mg/kg IV)
were given as a single dose in the first hour of the experiment as prophylaxis against
infection.
Heart rate (HR), oxygen saturation (Sa02, postductal), central venous pressure, rectal
temperature and systolic (SBP), diastolic (DBP) and mean arterial (MAP) blood pressures
were continuously monitored. An initial arterial blood gas (ABG) sample was drawn
•
immediately after placement of the arterialline with subsequent hourly sampling until the
end of the experiment (EBL 30 Acid-Base Analyzer, Copenhagen, Denmark). Inspired
30
oxygen (Fi02) was adjusted to maintain oxygen saturation greater than 90% and Pa02
greater than 70 mmHg. Ventilatory parameters were adjusted according to arterial blood
gases in order to maintain pH between 7.3 and 7.4 and a PC02 between 40 and 50
mmHg.
2. Selective Pulmonary Distention
A right posterolateral thoracotomy in the fifth interspace was performed. The right
upper lobe (RUL) bronchus was isolated and ligated at its tracheal junction with 3-0 silk
ties. The bifurcation into anterior and posterior segments occurred within millimeters of
the tracheal bronchus junction. Therefore, we had to use two double lumen 4Fr catheters
in order to cannulate each segment. Each catheter was held in place with 3-0 silk ties. The
distal lumen of each catheter was connected to a pressure monitor (Hewlett Packard). The
proximal lumen of each catheter was used to infuse pre-oxygenated perfluorocarbon
(PFC) until a pressure of 10 mmHg was reached. Additional PFC was added throughout
•
the experiment when the pressure feH below 7 mmHg. Both catheters were secured to the
skin with 3-0 silk and the thoracotomy incision was closed with running 3-0 silk.
Three hours prior to sacrifice, aH piglets received a single intravenous injection of
thymidine 3H (lmCi/kg) in order to determine the rates ofDNA synthesis post-mortem.
The operated control group was anaesthetized and ventilated for 12 hours and also
underwent a thoracotomy and dissection of their RUL bronchus without ligation or
perfluorocarbon distention. The non-operated control group only received the injection of
thymidine 3H three hours prior to sacrifice without any surgieal intervention or
experimental manipulation.
3. Neonatal piglet euthanasia and autopsy
At the end of the experimental period, while still under general anesthesia, aH
piglets were euthanized with an overdose of IV pentothal. Their chest was re-opened and
the lungs and heart were removed en bloc. The right and left upper bronchi were
dissected and ligated using 3-0 silk enabling isolation of both right and left upper lobes.
The posterior segmental bronchi to the right and left upper lobes were dissected and
31
ligated. The right posterior segment (RPS) and the left posterior segment (LPS) were then
removed and immediately frozen with dry ice and stored at -80°C.
D) MOLECULAR ANALYSIS
Both the RPS and the LPS were analyzed for their respective amount of total
DNA by fluorometry. Results were 'normalized' by dividing the total quantity of DNA
by the weight of each lung sample prior to homogenization. The rate of incorporation of
thymidine
3H
into DNA was determined from these homogenates. Although 5%
trichloroacetic acid precipitates aIl DNA within the homogenate, the scintillation counter
measures only the radioactivity emanating from the DNA with incorporated thymidine
3H.
Rates of DNA synthesis were then calculated by dividing the amount of incorporated
thymidine 3H (CPM) by the total amount of DNA for that particular lung segment (ng
DNA/mg tissue). The right and left upper lobes were compared to each other, with the
left upper lobe serving as a control. Of note, the above analyses were performed by Dr. B.
•
Piedboeuf and his assistant
St~phane
Guay (Universite Laval, Quebec, QC).
C) OUTCOME MEASURES
These were as follows:
1. Survival
2. Arterial blood gas trends: Pa02, PaC02, pH
3. Total DNA (in RPS or LPS) (ng DNA/mg tissue) = total quantity ofDNA
weight of sample (RPS or LPS)
4. Rate of DNA synthesis (in RPS or LPS) (CPM/ng DNA/mg tissue) = thymidine 3H incorporation
total DNA synthesis (RPS or LPS)
5. DifferentiallungDNA synthesis rate (%) = (Rate ofDNA synthesis in RPS) x 100
Rate of DNA synthesis in LPS
D) STATISTICAL ANALYSIS
We analyzed our results using the statistical software package SPSS (version
10.05). One-way ANOVA with Bonferonni or Dunnett's post-hoc testing was used to
compare right and 1eft lungs between the 4 experimental groups. Paired t-tests were used
32
to compare right and left lungs within each group. Survival was compared using a chi-
•
square test with Yates' correction. Significance occurred when p < 0.05 .
•
•
33
•
RESULTS
EXPERIMENT
#1:
FETAL
TRACHEAL
OCCLUSION
&
EXOGENOUS
SURFACTANT AT BIRTH IN A SURGICALLYCREATED CDH LAMB MODEL
Two groups of CDH lambs were created in this set of experiments: (i) CDH +
Surfactant (SURF) and (ii) CDH + TO + SURF and compared with 3 groups of lambs
previously resuscitated in our lab using an identical protocol (112): non-operated
controls, CDH and CDH + TO. The mortality rates for both sets of experiments were
23.5% and 34% respectively. Both rates are inferior to the commonly reported rate of
50% for fetal CDH lamb experiments (49).
Only animaIs with a diaphragmatic defect and hemiated viscera in the left chest at the
time of autopsy were considered as CDH ± TO ± SURF lambs. One animal was excluded
because of an inadequate CDH. Thus, the number of lambs analyzed per group were: (a)
CDH (n=5), (b) CDH + TO (n=5), (c) CDH + SURF (n=4), (d) CDH + TO + SURF
(n=6), (e) Controls (n=4).
AU 10 lambs from both SURF groups survived the 8 hour resuscitation period. In
contrast, only 3 of the 5 pure CDH lambs survived (Table 1). The incidence of chest tube
placement for the treatment of tension pneumothoraces was also recorded (Table 1).
None of the control animaIs required chest tubes. In contrast, all CDH only animaIs
required chest tubes while three of the five CDH + TO lambs had chest tubes placed. The
addition of exogenous surfactant appeared to decrease the incidence of pneumothoraces
although this was not stastically significant due to the small number of animaIs per group.
Three of the four CDH + SURF lambs required chest tubes while only 2 of the 6 CDH +
TO + SURF animaIs needed chest tubes.
Both CDH and CDH + SURF lungs were hypoplastic with wet lung weightlbody
weight (LWIBW) ratios of 1.11 ± 0.12% and 0.99 ± 0.14% respectively (Figure 1). The
addition of TO ± SURF significantly increased the LW/BW (2.39 ± 0.42% and 2.14 ±
0.23%) to that of controls (1.73 ± 0.04%) (Figure 2). Fetal TO resulted in doubling of
both right and left lung weights when compared with unoccluded CDH lungs (Table 2).
Proportional growth occurred in both lungs as manifested by similar right to left lung
•
ratios of CDH ± TO ± SURF (Table 2).
34
•
The dry-to-wet ratios, which reflect the amount of water within each set of lungs, was
similar for aIl Iambs (Figure 3). Dry LW/BW ratios paraIleled their wet LW/BW
counterparts, with increased dry lung weights in both TO groups compared to CDH ±
SURF groups (Figure 4).
Gas exchange, as measured by arterial pH, PaC02 and Pa02, was not significantly
improved with the addition of surfactant (Figures 5-7). Although improvements in pH
were observed after 180 minutes in CDH + SURF lambs compared with their CDH
counterparts, this was not significant. CDH lambs did have worsening pH (i.e. Iower,
more acidotic values) from 180 minutes to 360 minutes compared with controls and CDH
+ TO ± SURF (Figure 5). CDH + SURF, CDH + TO ± SURF and controls maintained
similar pH values over the 8 hour resuscitation. PaC02 values were similar over time
between CDH and CDH + SURF lambs (Figure 6). Significant differences were noted
beginning at 240 minutes when CDH ± SURF lambs had higher PaC02 leveis than
controis and CDH + TO lambs. The addition of surfactant to CDH + TO lambs did not
•
significantly improve PaC02. In fact, CDH + TO + SURF lambs demonstrated worsening
PaC02 after 240 minutes with levels approaching those of CDH ± SURF lambs for the
remaining 4 hrs of the resuscitation. In contrast, Pa02 was similar for an groups for the
entire 8 hours (Figure 7). Although CDH lambs had the lowest Pa02 levels after 180
minutes, this was not statisticaIly significant. Despite lower (i.e. improved) AaD02 from
240 minutes onwards in the CDH + SURF, CDH + TO ± SURF and control Iambs, this
trend was not significant (Figure 8).
Ease of ventilation, as measured by decreased MVI and increased VEl, did not
improve significantly with the addition of surfactant. MVI remained elevated throughout
the experiment in CDH lambs (Figure 9). The addition of surfactant did notsignificantly
decrease MVI. Both controis and CDH + TO lambs increased their VEI after 240 minutes
while CDH ± SURF and CDH + TO + SURF groups maintained low VEI values for the
duration of the experiment (Figure 10).
Airway pressure (Paw) was significantly 10wer in both surfactant groups with CDH +
TO + SURF consistently having the 10west airway pressures (Figure Il). Oxygenation
index (01), which incorporates Paw and Pa02, was not consistently significantly different
between the groups (Figure 12). However, an interesting trend was noted with CDH ±
35
•
SURF groups having higher ors after 180 minutes while CDH + TO ± SURF and
control groups tended to maintain similar ors throughout the resuscitation.
Pulmonary compliance was lowest for the CDH only group throughout the
resuscitation (Figure 13). The addition of surfactant significantly improved compliance.
Not only did the CDH + SURF group maintain higher compliance than their CDH
counterparts, they also achieved compliance similar to both control and CDH + TO
groups. Although CDH + TO + SURF lambs had the highest compliance values at aH
times, statistical significance occurred only in the first 4 hours of the experiment.
•
•
36
•
EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC
LIQUID IN HEALTHY, NEONATAL PIGLETS
Four groups of piglets were created in this set of experiments: (a) PFC distention x 6
hours (PFC-6), (b) PFC distention x 12 hours (PFC-12), (c) operated controis (OC) and
(d) non-operated controis (NOC). AlI piglets, except one, survived and completed the
experiment. Our only mortality was precipitated by mechanicai failure of the ventilator
shortly after tracheotomy and initiation of CMV. The ventilator failed to alarm, resulting
in delayed recognition of the mechanicai failure. Upon opening the piglet's chest, severe
bradycardia was noted and persisted despite the return of ventilator function. This piglet
was therefore immediately sacrificed with an intracardiac injection of pentothal and
excluded from analysis. Another piglet had intrabronchiai pressures less than 5 for over 2
hours despite repeated boluses of PFC liquid. Upon re-opening the chest, one of the
catheters was noted to have been dislodged from its segment. This piglet was
consequently also excluded from analysis. Thus, the number of piglets analyzed per
•
group were: (a) PFC-6 (n=lO), (b) PFC-12 (n=5), (c) operated controls (n=4) and (d) nonoperated controls (n=4). AlI piglets were similar in terms ofheart rate, oxygen saturation,
temperature, mean arterial pressure (MAP), pH, PaC02 and Pa02.
The total amount of DNA per lung segment (Figure 14), the rates of DNA synthesis
per lung segment (Figure 15) and the differential rates of DNA synthesis (Figure 16)
were determined from our molecular analysis. Both control groups had similar amounts
of total DNA in both their RPS and LPS. On the other hand, total DNA in the RPS was
significantly lower than LPS within the PFC-6 group (p<0.05) and close to significance
in the PFC-12 group (p=0.067). Operated controls had the highest total DNA content in
their RPS compared with aIl groups (Figure 14). However, this was only significantly
different when compared with the total DNA content in the RPS for the PFC-6 group. On
the other hand, non-operated controls had significantly lower total DNA content in their
LPS compared with the OC and PFC-12 groups. In contrast, rates of DNA synthesis per
lung segment were similar within each group and between groups (Figure 15). FinaIly,
although the differential rate of DNA synthesis was statisticaIly similar between aIl 4
•
groups, the PFC-12 piglets had the overaIl highest rate (202% vs. 145% (PFC-6), 126%
(OC) and 162% (NOC) (Figure 16).
37
CONCLUSION
EXPERIMENT #1: FETAL TRACHEAL OCCLUSION & EXOGENOUS
SURFACTANT AT BIRTH IN A SURGICALLY CREATED CDH LAMB MODEL
As expected, only fetal Ta, and not the addition of surfactant, resu1ted in reversaI
of pulmonary hypoplasia. A1though the exact mechanism of action of Ta remains
unknown, it is believed that increases in intratracheal pressure and lung liquid volume
stimulate alveolar and epithelial cell proliferation via stretch-induced mechanisms (26,
28, 62-63). Accelerated lung growth continues as long as Ta is maintained. The degree
oflung growth is dependent on the length of the occlusion: lambs with Ta for only 1 or 2
weeks experience much less growth than those with Ta for 3 weeks (29,64). In addition,
Ta prevents excess pulmonary muscularization which decreases pulmonary hypertension
at birth, leading to improved gas exchange, ventilation and compliance (68-69).
Unfortunately, Ta accelerates lung growth at the expense of type II cells (29).
Tracheal release (TR) has been advocated as a means of preventing this decrease in type
II pneumocyte density. Prior work in our laboratory has demonstrated recovery of type II
cells to control levels with TR performed 2 days prior to delivery in normal lambs (71)
and 1 week prior to delivery in surgically created CDH lambs (72). However, surfactant
levels remained low in the CDH + Tü ± TR animaIs, suggesting that exogenous
surfactant at birth may be beneficial.
In the present study, we have demonstrated that prophylactic surfactant does not
improve gas exchange nor ventilation over an 8 hour resuscitation period in CDH lambs.
In fact, Paca2 worsened after 240 minutes in the CDH + Ta + SURF group. This may be
a consequence of both the volume of surfactant given and its method of administration.
The second dose of surfactant was calculated based on the lamb's body weight rather
than lung weight, resu1ting in an overestimation of the amount of surfactant required.
Consequently, this second dose may have 'drowned' the lungs, rendering gas exchange
and ventilation more difficult. In addition, the latter dose of surfactant required manual
bagging using less than 100% oxygen and inconsistent PIP. Even though this dose was
rapidly administered over 3-5 minutes, the lungs of these CDH lambs appear to be very
sensitive to even small amounts of suboptimal oxygenation and ventilation. The CDH +
Ta + SURF lambs failed to recover after the 2nd dose of surfactant and continued to
38
•
demonstrate high PaC02 levels for the remaining 4 hours of the resuscitation. In contrast,
the CDH + TO group had significantly lower PaC0 2 levels after 240 minutes compared
with CDH + SURF lambs. Thus, exogenous surfactant alone is inferior to TO with
regards to improving hypercarbia. Prophylactic surfactant did not improve oxygenation
since aIl five groups maintained similar Pa02 levels during the entire 8 hour experiment.
Similarily, ventilation was not improved with the addition of surfactant. CDH ± SURF
lambs maintained similar ventilation values throughout the experiment. On the other
hand, CDH + TO + SURF lambs failed to experience improvements in their MVI and
VEI values in the latter half of the experiment compared with their CDH + TO
counterparts. Both MVI and VEI incorporate respiratory rate (RR), peak inspiratory
pressures (PIP) and PaC02 levels. Despite lower PIP levels in the CDH + TO + SURF
group, the significantly higher PaC02 levels in the last 4 hours of the experiment
presumably led to more difficult ventilation in this group.
In contrast, airway pressure (Paw) remained significantly lower in both surfactant
•
groups compared with their respective counterparts. Pulmonary compliance was also
significantly improved in both surfactant groups, especially the CDH + SURF group
which maintained compliance levels similar to control and CDH + TO groups.
Presumably, these changes in both Paw and compliance reduced pulmonary barotrauma,
resulting in fewer tension pneumothoraces. Although exogenous surfactant has been
argued as predisposing patients to an increased incidence of pneumothoraces, a recent
meta-analysis of randomized, controlled trials demonstrated a decreased incidence of
pneumothorax in premature infants with RDS receiving prophylactic surfactant (92). In
this study, our data also trended towards a decreased incidence of tension
pneumothoraces.
Oxygenation index (OI), which normally incorporates postductal Pa02, mean
airway pressure and Fi02, estimates the degree of shunting occurring in the CDH
neonate. OI is used in the clinical setting to assess the severity of respiratory failure, to
predict the rate of mortality without ECMO and as a selection criteria for ECMO (2).
Although our OI is based on Paw and preductal Pa02 which prevents us from correlating
•
known parameters as an estimation of survival, an interesting trend was noted with CDH
± SURF lambs increasing their OI in the latter half of the experiment. Thus, it appears
39
•
that these lambs suffered from worse respiratory function than their control and CDH ±
TO ± SURF counterparts.
Our results contradict similar experiments performed by colleagues in Buffalo
who observed that CDH + SURF lambs had improved Pa02, decreased PaC02 and
increased pH compared with their CDH counterparts. However, their longest
resuscitation was only 4 hours and no control groups were used as comparisons (85-86).
Although we used bovine lipid extract surfactant (BLES) rather than calf lung surfactant
(Infasurf), both are natural surfactants which are considered supenor to synthetic
surfactants. Natural surfactant preparations contain surfactant proteins without the
addition of detergents to decrease alveolar surface tension (30). They have been shown to
be more effective in the treatment of RDS than their synthetic counterparts (113-115).
Similar comparison studies for respiratory failure associated with CDH have not been
performed.
Our results clearly demonstrate that prophylactic surfactant in a surgically created
•
CDH lamb model provides no added benefit in terms of gas exchange and ventilation but
does improve compliance. CDH + TO lambs were less acidotic, hypercarbic and easier to
ventilate than CDH + SURF lambs, suggesting that surfactant deficiency appears to play
a less important role in the pathophysiology of CDH. However, the marked
improvements in compliance observed with the CDH + SURF lambs justifies the
administration of prophylactic surfactant in cases where CDH is diagnosed prenatally but
in whom TO is either not required, contraindicated or refused by the parents. This
increase in compliance could decrease pulmonary barotrauma and the incidence of
tension pneumothoraces. In addition, the improved compliance observed in the CDH +
TO and CDH + SURF groups to levels approaching the CDH + TO + SURF group
midway into the resuscitation, suggests that the lamb's endogenous surfactant system
begins to play a more important role around 4 hours of life. Similar improvements in pH
and PaC02 in the CDH + TO lambs compared with the CDH ± SURF groups after 4
hours provides further evidence for delayed in vivo surfactant secretion. Regulation of
endogenous surfactant remains poorly understood and further studies are required.
•
Fetal TO continues to yield the best results in terms of overall postnatal lung
function, likely acting via surfactant independent mechanisms. Prenatal glucocorticoids
40
•
have also been shown to improve lung function without the help of surfactant (76-77).
This improvement in lung function is attributed to accelerated prenatal lung growth and
increased pulmonary artery remodeling. Survival of CDH neonates receiving extracorporeal membrane oxygenation (ECMO) requires a minimum lung volume of 45%
compared with age-matched controls (42). Pulmonary artery remodeling, by altering the
collagen:elastin ratio and decreasing alveolar wall thickness, results in greater alveolar
distensibility which leads to increased lung compliance. In this study, accelerated
prenatal lung growth and reversaI of pulmonary hypoplasia, rather than repletion of
surfactant levels at birth, appear to play a more important role in improving postnatal
lung function in surgically created CDH lambs. AlI of our results support the hypothesis
that the most important factor in CDH is pulmonary hypoplasia and therefore methods to
reverse pulmonary hypoplasia, such as TO and antenatal steroids, yield significantly
better results. Other interventions which do not treat pulmonary hypoplasia, such as
exogenous surfactant, are unlikely to be useful when administered alone. However,
•
surfactant therapy can be a useful adjunct in the treatment of CDH.
EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC
LIQUID IN HEALTHY, NEONATAL PIGLETS
Turning to our second set of experiments involving PFC distention of the RUL in
healthy piglets, we elected to measure the rate of DNA synthesis within the distended
pulmonary segment as an indirect method of measuring accelerated postnatal lung
growth. This method was chosen since the small amount of lung tissue used and the short
duration of the experiment precluded any weight-based analysis. Unlike Nobuhara's
group (97), we were unable to demonstrate increased rates of DNA synthesis after PFC
distention. Our negative results may be due to several factors. Technical difficulties, both
during the surgery and with the molecular analyses, were at the forefront of these
experiments. Several animaIs developed subpleural collections of PFC, either after the
initial bolus or during subsequent boluses of PFC. These collections compressed the
adjacent lung tissue rather than distending it, decreasing the amount of stretch by the PFC
•
liquid within the lung parenchyma. However, even after repeating the data analysis
without these animaIs, rates of DNA synthesis remained similar between groups. Thus,
41
•
this was not the only factor adversely affecting our results. The tests used in our
molecular analysis were only recently developed. The sensitivity, reliability and validity
of these tests remains to be determined. Thymidine 3H was injected only 3 hours prior to
sacrifice, rather than 8 hours as other researchers have reported (60). Consequently, the
number of dividing cells within this 3 hour time period is much less than the total number
of cells within the lung segment being studied. Our moiecular tests may not have been
sensitive enough to detect these small changes in cell number. We are currently
examining other techniques to more accurately quantify the rates of DNA synthesis
within pulmonary tissue. Finally, and most importantIy, our experiments were probably
of insufficient duration. We know from fetai TO studies in sheep that markedIy increased
rates of DNA synthesis occur around 48 hours (60). Nobuhara (97) demonstrated
increased DNA synthesis after 21 days of continuous PFC distention in the RUL of
sheep. It would be interesting to analyze lung tissue at various time points (i.e. 24 hours,
48 hours, 72 hours etc.) to determine exactly when this acceleration in DNA synthesis
•
takes place.
Finally, our observation that the total amount of DNA was markedly less in the
RPS than the LPS in both PFC groups was unexpected (Figure 14). We believe that
residual amounts of PFC liquid in the lung sample may have falsely increased the
sample's weight, resulting in less total DNA calculated per segment. If we had used a
third lung segment to calculate wet-to-dry ratios, we would have obtained the dry weight
of RPS and LPS. These calculations wouid have enabled us to determine if residual PFC
artificially decreased the total amount of DNA per segment. Further studies remain to be
performed before any definitive conclusions can be reached.
•
42
•
SUMMARY
In our CDH lamb model, we demonstrated that fetal TO accelerated prenatallung
growth which led to improvements in postnatal lung function. Despite the addition of
exogenous surfactant at birth, gas exchange and ventilation were not improved. However,
compliance was markedly increased after surfactant administration. This improvement in
compliance, in combination with a reduction in airway pressure, decreased pulmonary
barotrauma as manifested by a reduction in the incidence of tension pneumothoraces.
Although fetal TO alone yields the best results in terms of overall lung function, the use
of prophylactic surfactant is justified in prenatally diagnosed CDH neonates in whom
fetal surgery is either contraindicated or refused. Surfactant independent mechanisms,
such as increased lung growth and pulmonary arterial growth and remodeling, are
believed to be responsible for the improvements in postnatallung function after fetal TO.
In our piglet model, PFC distention of the RUL failed to accelerate postnatal lung
growth. Technical difficulties and the short duration of the experiments contributed to
•
these negative results.
43
•
REFERENCES
1. Katz AL, Wisewell TE, Baumgart S: Contemporary controversies in the management
of congenital diaphragmatic hernia. Clin Perinatol 25 :219-248, 1998
2. Arensman Rm and Bambini DA: Congenital diaphragmatic hernia and eventration. In:
Ashcraft KW et al. Pediatrie Surgery 3rd edition, Philadelphia, W.B. Saunders Company,
300-317,2000
3. Wilcox DT, Irish MS, Holm BA, et al: Prenatal diagnosis of congenital diaphragmatic
hernia with predictors ofmortality. Clin PerinatoI23:701-709, 1996
4. Guibaud L, Filiatrault D, Garel L, et al: Fetal congenital diaphragmatic hernia:
accuracy of sonography in the diagnosis and prediction of the outcome after birth. AlR
Am 1 RoentgenoI166:1195-1202, 1996
5. Langham MR, Kays DW, Ledbetter Dl, et al: Congenital diaphragmatic hernia:
epidemiology and outcome. Clin Perinatol 23 :671-688, 1996
6. Harrison MR, Adzick NS, Estes lM, et al: A prospective study of the outcome for
•
fetuses with diaphragmatic hernia. lAMA 271:382-384, 1994
7. Cartlidge PH, Mann NP, Kapila L: Pre-operative stabilisation in congenital
diaphragmatic hernia. Arch Dis Child 61: 1226-1228, 1986
8. Langer l, Filler R, Boho D, et al: Timing of surgery for congenital diaphragmatic
hernia: 1s emergency operation necessary? 1 Pediatr Surg 23 :731-734, 1988
9. Charlton A, Bruce l, Davenport M: Timing of surgery in congenital diaphragmatic
hernia: Low mortality after pre-operative stabilization. Anaesthesia 46:820-823, 1991
10. Coughlin l, Drucker D, Cullen M, et al: Delayed repair of congenital diaphragmatic
hernia. Am Surg 59:90-93, 1993
11. Wilson lM, Lund DP, Lillehei CW, et al: Delayed repair and preoperative ECMO
does not improve survival in high-risk congenital diaphragmatic hernia. 1 Pediatr Surg
27:368-372, 1992
12. The Neonatal 1nhaled Nitric Oxide Study Group: 1nhaled nitric oxide and hypoxic
respiratory failure in infants with congenital diaphragmatie hernia. Pediatries 99:838-845,
1997
•
44
•
13. Breaux C Jr, Rouse T, Cain W, et al: Improvement in survival of patients with
congenital diaphragmatic hemia utilizing a strategy of delayed repair after medical and/or
extracorporeal membrane oxygenation stabilization. J Pediatr Surg 26:333-338, 1991
14. Adolph V, Flageole H, Perreault T, et al: Repair of congenital diaphragmatic hemia
after weaning from extracorporeal membrane oxygenation. J Pediatr Surg 30:349-352,
1995
15. Heiss K, Clark R: Prediction of mortality in neonates with congenital diaphragmatic
hemia treated with extracorporeal membrane oxygenation. Crit Care Med 23:1915-1919,
1995
16. Wilson J, Lund D, Lillehei C, et al: Congenital diaphragmatic hemia - a tale oftwo
cities: The Boston experience. J Pediatr Surg 32:401-405, 1997
17. Frenckner B, Ehren H, Granholm T, et al: Improved results in patients who have
congenital diaphragmatic hemia using preoperative stabilization, extracorporeal
membrane oxygenation, and delayed surgery. J Pediatr Surg 32:1185-1189, 1997
•
18. Shanley CJ, Hirschl RB, Schumacher RE, et al: Extracorporeal life support for
neonatal respiratory failure. A 20-year experience. Ann Surg 220:269-280, 1994
19. Metkus AP, Filly RA, Stringer MD, et al: Sonographic predictors of survival in fetal
diaphragmatic hernia. J Pediatr Surg 31: 148-152, 1996
20. Lipshutz GS, Albanese CT, Feldstein VA, et al: Prospective analysis of lung-to-head
ratio predicts survival for patients with prenatally diagnosed congenital diaphragmatic
hemia. J Pediatr Surg 32:1634-1638, 1997
21. Harrison MR, Adzick NS, Flake AW et al: Correction of congenital diaphragmatic
hemia in utero. VI: Hard-eamed lessons. J Pediatr Surg 28:1411-1418,1993
22. Harrison MR, Adzick NS, Bullard KM, et al: Correction of congenital diaphragmatic
hemia in utero VII: A prospective trial. J Pediatr Surg 32:1637-1642, 1997
23. Hedrick MH, Estes JM, Sullivan KM, et al: Plug the lung until it grows (PLUG): A
new method to treat congenital diaphragmatic hemia in utero. J Pediatr Surg 29:612-617,
1994
24. VanderWall KJ, Bruch SW, Meuli M, et al: Fetal endoscopie ("Fetendo") tracheal
clip. J Pediatr Surg 31 :1102-1103, 1996
45
•
25. Flageole H, Evrard VA, Vandenberghe K, et al: Tracheoscopie endotracheal
occlusion in the ovine model: Technique and pulmonary effects. J Pediatr Surg 32: 13281331, 1997
26. Nardo L, Hooper SB, Harding R: Lung hypoplasia can be reversed by short term
obstruction of the trachea in fetal sheep. Pediatr Res 38:690-696, 1995
27. Benachi A, Dommergues M, Delezoide AL, et al: Tracheal obstruction in
experimental diaphragmatic hemia: An endoscopie approach in the fetal lamb. Prenat
Diagn 17:629-634, 1997
28. Hashim E, Laberge J-M, Chen M-F, et al: Reversible tracheal obstruction in the fetal
sheep: effect on tracheal fluid pressure and lung growth. J Pediatr Surg 30:1172-1177,
1995
29. Piedboeuf B, Laberge J-M, Ghituleseu G, et al: Deleterious effect of tracheal
obstruction on type II pneumocytes in fetal sheep. Pediatr Res 41 :473-479, 1997
30. McCabe AJ, Wilcox DT, Holm BA, et al: Surfactant - a review for pediatrie
•
surgeons. J Pediatr Surg 35:1687-1700, 2000
31. Thurlbeek WM: Prematurity and the developing lung. Clin Perinatol 19:497-519,
1992
32. Jeffery PK: The development of large and smal1 airways. Am J Respir Crit Care Med
157:S174-S180, 1998
33. Alcom DG, Adamson TM, Maloney JE, et al: A morphologie and morphometric
analysis offetallung development in the sheep. Anat Ree 201 :655-667, 1981
34. Popper CG, Thurlbeek WM: Growth, Aging and Adaptation. In: Murray, JF and
Nadel JA, Textbook of Respiratory Medicine, 2nd edition, Philadelphia, W.B. Saunders
Company, 37-47, 1994
35. Lipsett J, Cool JC, Runeiman SC, et al: Morphometric analysis of pulmonary
development in the sheep fol1owing creation of fetal diaphragmatic hemia. Pediatr Pathol
Lab Med 17:789-807, 1997
36. Clements LP: Embryonie development of the respiratory portion of the pig's lung.
Anat Ree 70:575-590, 1938
•
37. Marrable AW: The embryonic pig: A ehronologieal aceount. London, Pitman, 1971
46
38. Baskerville A: Histological and ultrastructural observations on the development of the
lung of the fetal pig. Acta Anat 95:218-233, 1976
39. Moore KL and Persaud TVN: Body cavities, primitive mesenteries, and the
diaphragm, in Moore KL, Persaud TVN (eds): The Developing Human: Clinically
Oriented Embryology. 5th ed. Philadelphia, PA, W.B. Saunders Company, 1993, pp 174185
40. Kluth D, Losty PD, Schnitzer JJ et al: Toward understanding the developmental
anatomy of congenital diaphragmatic hemia. Clin PerinatoI23:655-669, 1996
41. Adzick NS, Outwater KM, Harrison MR, et al: Correction of congenital
diaphragmatic hemia in utero IV. An early gestational fetal lamb model for pulmonary
vascular morphometric analysis. J Pediatr Surg 20:673-680, 1985
42. Thibeault DW, Haney B: Lung volume, pulmonary vasculature, and factors affecting
survival in congenital diaphragmatic hemia. Pediatries 101 :289-295, 1998
43. Dillon E, Renwick M: Antenatal detection of CDH: The northern region experience.
•
Clin RadioI48:264-267, 1993
44. Nakamura Y, Harada K, Yamamoto l, et al: Human pulmonary hypoplasia. Arch
Pathol Lab Med 116:635-642, 1992
45. Pringle KC, Turner JW, Schofield JC, et al: Creation and repair of diaphragmatic
hernia in the fetallamb: Lung development and morphology. J Pediatr Surg 19:131-140,
1984
46. Berk C, Grundy M: High risk lecithin/sphingomyelin ratios associated with neonatal
diaphragmatic hemia. Case reports. Br J Obstet GynaecoI89:250-251, 1982
47. Hirthler MA, Bradley CA, Willis D, et al: The measurement of amniotic fluid
phospholipids in congenital diaphragmatic hernia. Pediatr Surg lnt (in press)
48.
Hisanga
S,
Shimokawa
H,
Kashiwabara
Y,
et
al:
Unexpected
low
lecithin/sphingomyelin ratios associated with fetal diaphragmatic hernia. Am J Obstet
Gynecol 149:905-906, 1984
49. Glick PL, Stannard VA, Leach CL, et al: Pathophysiology of congenital
diaphragmatic hernia II: The fetallamb CDH model is surfactant deficient. J Pediatr Surg
27:383-388, 1992
47
•
50. Wilcox DT, Glick PL, Karamanoukian RG, et al: Pathophysiology of congenital
diaphragmatic
hemia
XII:
Amniotic
fluid
lecithinlsphingomyelin
ratio
and
phosphatidylglycerol concentrations do not predict surfactant status in congenital
diaphragmatic hemia. J Pediatr Surg 30:410-412, 1995
51. Wilcox DT, Irish MS, Holm BA, et al: Animal models in congenital diaphragmatic
hemia. Clin PerinatoI23:813-822, 1996
52. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal trachealligation reverses
the structural and physiological effects of pulmonary hypoplasia in congenital
diaphragmatic hemia. J Pediatr Surg 29:248-257, 1994
53. DiMaio M, Ting A, Gil J, et al: A morphometric study oflung hypoplasia in a second
trimester lamb model of congenital diaphragmatic hemia. Am J Resp Crit Care Med
149:A727, 1994
54. Wilcox DT, Glick PL, Karamanoukian H, et al: Pathophysiology of congenital
diaphragmatic hemia V. Effect of exogenous surfactant therapy on gas exchange and
•
lung mechanics in the lamb congenital diaphragmatic hemia mode!. J Pediatr 124:289293, 1994
55. Ohkawa H, Matsumuto M, Hori T, et al: Familial congenital diaphragmatic hemia in
the pig - studies on pathologyand heredity. Eur J Pediatr Surg 3:67-71, 1993
56. Wigglesworth J, Hislop A: Fetallung growth in congenitallaryngeal atresia. Pediatr
Pathol 7:515-525, 1987
57. Alcom D, Adamson TM, Lambert TH: Morphologic effects of chronic tracheal
ligation and drainage in the fetallamb lung. Journal of Anatomy 123:649-660, 1977
58. Harrison MR, Mychaliska GB, Albanese CT, et al: Correction of congenital
diaphragmatic hemia in utero IX: fetuses with poor prognosis (liver herniation and low
lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion. J Pediatr
Surg 33:1017-1023, 1998
59. Wilson JM, DiFiore JW, Peters CA: Experimental fetaI trachealligation prevents the
pulmonary hypoplasia associated with fetal nephrectomy: possible application for
congenital diaphragmatic hernia. J Ped Surg 28:1433-1440, 1993
48
•
60. Nardo L, Hooper S, Harding R: Stimulation of lung growth by tracheal obstruction in
fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res 43: 184-190,
1998
61. Harrison MR, Adzick N, Flake A, et al: Correction of congenital diaphragmatic
hemia in utero. VIII: Response of the hypoplastic lung to tracheal occlusion. J Pediatr
Surg 31:1339-1348,1996
62. Kitano Y, Flake AW, Quinn TM, et al: Lung growth induced by tracheal occlusion in
the sheep is augmented by airway pressurization. J Pediatr Surg 35:216-222, 2000
63. Nobuhara KK, Wilson JM: The effect ofmechanical forces on in utero lung growth in
congenital diaphragmatic hemia. Clin Perinatol 23:741-751, 1996
64. Wild YK, Piasecki GJ, De Paepe ME, et al: Short-term tracheal occlusion in fetal
lambs with diaphragmatic hemia improves lung function, even in the absence of Iung
growth. J Pediatr Surg 35:775-779, 2000
65. Papadakis K, Luks FI, DePaepe M, et al: Fetallung growth after trachealligation is
•
not soIeIy a pressure phenomenon. J Pediatr Surg 32:347-351, 1997
66. Quinn TM, Sylvester KG, Kitano Y, et al: TGF-B2 is increased after fetai tracheal
occlusion. J Pediatr Surg 34:701-705, 1999
67. Nobuhara KK, DiFiore JW, Ibla JC, et al: Insulin-like growth factor-I gene
expression in three models of accelerated lung growth. J Pediatr Surg 1057-1061, 1998
68. Kanai M, Kitano Y, Radu A, et al: Effect of prenatal tracheal occlusion on pulmonary
arteriai structural changes in rats with nitrofen-induced congenital diaphragmatic hemia.
Surgical Forum L:567-568, 1999
69. Bratu 1, Flageole HF, Laberge J-M, et al: Pulmonary structural maturation and
pulmonary artery remodeling after reversible fetal ovine tracheal occlusion in
diaphragmatic hemia. J Pediatr Surg 36:739-744, 2001
70. Wirtz HR, Dobbs LG: The effects of mechanical forces on lung function. Resp
PhysioI1l9:1-17,2000
71. Kay S, Laberge J-M, Flageole H, et al: The use of antenatal steroids to counteract the
negative effects of tracheal occlusion in the fetallamb model. Pediatr Res (in press)
•
49
•
72. Bratu l, Flageole H, Laberge JM, et al: Surfactant levels after reversible tracheal
occlusion and prenatal steroids in experimental diaphragmatic hernia. J Pediatr Surg
36:122-127,2001
73. Rebello CM, Ikegami M, Polk DH, et al: Postnatal lung response and surfactant
function after fetal or maternaI corticosteroid treatment. J Appl Physiol 80:1674-1680,
1996
74. Ikegami M, Polk D, Jobe A: Minimum interval from fetal betamethasone treatment to
postnatallung responses in preterm lambs. Am J Obstet GynecoI174:1408-1413, 1996
75. Jobe AH, Polk D, Ikegami M, et al: Lung responses to ultrasound-guided fetal
treatments with corticosteroids in preterm lambs. J Appl Physiol 75:2099-2105, 1993
76. Fiascone JM, Jacobs HC, Moya FR, et al: Betamethasone increases pulmonary
compliance in part by surfactant-independent mechanisms in preterm rabbits. Pediatr Res
22:730-735, 1987
77. Beck JC, Mitzner W, Johnson JWC, et al: Betamethasone and the rhesus fetus: Effect
•
on lung morphometry and connective tissue. Pediatr Res 15:235-240, 1981
78. Pinkerton KE, Willet KE, Peake JL, et al: Prenatal glucocorticoid and T4 effects on
lung morphology in preterm lambs. Am J Respir Crit Care Med 156:624-630, 1997
79. Hedrick HL, Kaban JM, Pacheco BA, et al: Prenatal glucocorticoids improve
pulmonary morphometrics in fetal sheep with congenital diaphragmatic hernia. J Pediatr
Surg 32:217-222, 1997
80. Suen HC, Bloch KD, Donahoe PK: Antenatal glucocorticoid corrects pulmonary
immaturity in experimentally induced congenital diaphragmatic hernia in rats. Pediatr
Res 35:523-529, 1994
81. Ballard PL, Ballard RA: Scientific basis and therapeutic regimens for use of antenatal
glucocorticoids. Am J Obstet Gynecol 173:254-262, 1995
82. Suen HC, Catlin EA, Ryan DP: Biochemical immaturity of lungs in congenital
diaphragmatic hernia. J Pediatr Surg 28:471-475, 1993
83. Ijsselstijn HI, Zimmerman LJI, Bunt JEH, et al: Prospective evaluation of surfactant
composition in bronchoalveolar lavage fluid of infants with congenital diaphragmatic
•
hernia and of age-matched controls. Crit Care Med 26:573-580, 1998
50
84. Kendig JW, Notter RH, Cox C, et al: A comparison of surfactant as immediate
prophylaxis and as rescue therapy in newboms of less than 30 weeks gestation. N Engl J
Med 324:865-871, 1991
85. O'Toole SJ, Karamanoukian HL, Morin III FC, et al: Surfactant decreases pulmonary
vascular resistance and increases pulmonary blood flow in the fetal lamb model of
congenital diaphragmatic hemia. J Pediatr Surg 31 :507-511, 1996
86. Karamanoukian HL, Glick PL, Wilcox DT, et al: Pathophysiology of congenital
diaphragmatic hemia VIII: Inhaled nitric oxide requires exogenous surfactant therapy in
the lamb model of congenital diaphragmatic hemia. J Pediatr Surg 30: 1-4, 1995
87. Glick PL, Leach CL, Besner GE, et al: Pathophysiology of congenital diaphragmatic
hemia III: Exogenous surfactant therapy for the high-risk neonate with CDH. J Pediatr
Surg 27:866-869, 1992
88. O'Toole SJ, Karamanoukian HL, Irish MS, et al: Trachealligation: the dark side of in
utero congenital diaphragmatic hemia treatment. J Pediatr Surg 32:407-410, 1997
•
89. O'Toole SJ, Karamanoukian HL, Sharma A, et al: Surfactant rescue in the fetallamb
model of congenital diaphragmatic hemia. J Pediatr Surg 31: Il 05-11 09, 1996
90. Lotze A, Knight GR, Anderson KD: Surfactant (beractant) therapy for infants with
congenital diaphragmatic hemia on ECMO: Evidence of persistent surfactant deficiency.
J Pediatr Surg 29:407-412,1994
91. Bos AP, Tibboel D, Hazebroek FWJ, et al: Surfactant replacement therapy in highrisk congenital diaphragmatic hemia. Lancet 338:1279, 1991
92. SolI RF, Morley CJ: Prophylactic versus selective use of surfactant for preventing
morbidity and mortality in preterm infants. Cochrane Database Syst Rev 2:CD000510,
2000
93. Clark LC: Introduction. Fed Proc 29:1698, 1970
94. Schaffer TH, Wolfson MR, Clark LC: Liquid ventilation. Pediatr Pulmonol 14:102109, 1992
95. Day SE, Gedeit RG: Liquid ventilation. Clin PerinatoI25:711-722, 1998
96. Clark LC, Gollan F: Survival of mammals breathing organic liquids equilibrated with
oxygen at atmospheric pressure. Science 152: 1755-1756, 1966
51
97. Nobuhara KK, Fauza DO, DiFiore JW et al: Continuous intrapulmonary distention
with perfluorocarbon accelerates neonatal (but not adult) lung growth. J Pediatr Surg
33 :292-298, 1998
98. Major D, Cadenas M, Cloutier R, Fournier L, Wolfson MR, Shaffer TH: Combined
gas ventilation and perfluorochemical tracheal instillation as an alternative treatment for
lethal congenital diaphragmatic hernia in lambs. J Pediatr Surg 30: 1178-1182, 1995
99. Wilcox DT, Glick PL, Karamanoukian HL, et al: Pertluorocarbon-associated gas
exchange improves pulmonary mechanics, oxygenation, ventilation, and allows nitric
oxide delivery in the hypoplastic Iung congenital diaphragmatic hernia lamb mode!. Crit
Care Med 23:1858-1863, 1995
100. Pranikoff T, Gauger PG, Hirschl RB: Partialliquid ventilation in newborn patients
with congenital diaphragmatic hernia. J Pediatr Surg 31 :613-618, 1996
101. Nobuhara KK, Ferretti ML, Siddiqui AM et al: Long term effect of perfluorocarbon
distention on the lung. J Pediatr Surg 33: 1024-1028, 1998
•
102. de Luca U, Cloutier R, Laberge J-M et al: Pulmonary barotrauma in congenital
diaphragmatic hernia: Experimental study in lambs. J Pediatr Surg 22:311-316, 1987
103. Evrard VA, Verbreken EA, Vandenberghe K, Lerut, T, Flageole H, Deprest JA:
Endoscopie in utero tracheal plugging in the fetallamb to treat congenital diaphragmatic
hernia. J Am Assoc Gynecol Laparsoc 3:S11, 1996
104. Flageole H, Evrard VA, PiedboeufB, Laberge JM, Lerut TE, Deprest JA: The plugunplug sequence: an important step to achieve type II pneumocyte maturation in the fetai
lamb mode!. J Pediatr Surg 33:299-303, 1998
105. Deprest JAM, Evrard VA, Van Baillaer PP et al: Tracheoscopie endoluminal
plugging using an inflatabledevice in the fetallamb mode!. Eur J Obstet Gynecol Reprod
Biol 81:165-169, 1998
106. Jenkin G, Jorgensen G, Thorburn GD: Induction of premature delivery in sheep
following infusion of cortisol in the fetus. 1. The effect of maternaI administration of
progestagens. Can J Physiol PharmacoI63:500-508, 1985
107. Ballard PL, Ning Y, Polk D et al: Glucocorticoid regulation of surfactant
•
components in immature lambs. Am J PhysioI273:LI048-LI057, 1997
52
•
108. Ikegami M, Polk DH, Jobe AH, Newnham J, Sly P, Kohen R, Kelly R: Postnatal
lung function in 1ambs after fetal hormone treatment. Effects of gestational age. Am J
Respir Crit Care Med 152:1256-1261, 1995
109. Schnitzer 11, Hedrick HL, Pacheco BA, Losty PD, Ryan DP, Doody DP, Donahoe
PK: Prenatal glucocorticoid therapy reverses pulmonary immaturity in congenital
diaphragmatic hernia in fetal sheep. Ann Surg 224: 430-437, 1996
110. Beek JC, Mitzner W, Johnson JWC, et al: Betamethasone and the rhesus fetus:
Effect on lung morphometery and connective tissue. Pediatr Res 15: 235-240, 1981
111. Stolar CJH and Dillon PW: Congenital diaphragmatic hemia and eventration. In:
O'Neill JA et al. Pediatric Surgery 5th edition, St. Louis, Missouri, Mosby-Year Book,
Inc., 819-837, 1998
112. Bratu I: Lung growth, structural remodeling, surfactant levels, and 1ung function
after reversible tracheal occlusion in congenital diaphragmatic hemia. Masters of Science
Thesis submitted to McGill University, Montreal, QC, Canada, June 2000
•
113. Network V-ON: A multicenter, randomized trial comparing synthetic surfactant with
modified bovine surfactant extract in the treatment of neonatal respiratory distress
syndrome. Pediatries 97: 1-6, 1996
114. Hudak ML, Martin DJ, Egan EA, et al: A multicenter randomized masked
comparison trial of synthetic surfactant versus calf lung extract in the prevention of
neonata1 respiratory distress syndrome. Pediatrics 100:39-50, 1997
115. B100m BT, Kartwinkel J, Hall RT, et al: Comparison of Infasurf (Calf Lung
Surfactant Extract) to Survanta (Beractant) in the treatment and prevention of respiratory
distress syndrome. Pediatrics 100:31-38, 1997
•
53
•
TABLES
EXPERIMENT #1:
Table 1: Survival and Incidence of Chest Tubes
GROUP
AGEATDEATH
SURVIVED
CHESTTUBE
(HOURS)
8 HOURS
CDH
3/5
5, 7
5/5
CDH+SURF
4/4
nia
3/4
CDH+TO
5/5
nia
3/5
CDH + TO + SURF
6/6
nia
2/6
CONTROL
4/4
nia
0/4
•
54
•
Table 2: Right and Left Lung Growth
GROUP
RLW/BW(%)
LLW/BW(%)
RLWILLW
CDH
0.79 ± 0.08
0.32 ± 0.04
2.51 ± 0.18
CDH+TO
1.66 ± 0.23*
0.69 ± 0.18
2.78 ± 0.44 t
CDH+SURF
0.57 ± 0.34
0.23 ± 0.02
2.48 ± 0.13
CDH + TO + SURF
1.36 ±0.17*
0.70 ± 0.07*
1.90 ± 0.09
CONTROL
1.05 ± 0.38
0.66 ± 0.02*
1.59 ± 0.08
Legend:
RLW = right lung weight, LLW = left lung weight, BW = body weight
Data are shown as mean ± SEM where
*=
different from CDH ± SURF;
from control (p<0.05).
•
55
t
=
different
FIGURES
Figure 1: The Pathophysiology of CDH
An illustration of the development of acute respiratory failure in neonates with CDH.
EXPERIMENT #1:
Figure 2: Wet Lung Weight / Body Weight
Data is presented as mean ± SEM where *=different from CDH ± SURF (p<O.OS).
Figure 3: Dry-to-Wet Lung Weight Ratios
Data is presented as mean ± SEM. No significant differences exist between the S groups.
Figure 4: Dry Lung Weight / Body Weight
Data is presented as mean ± SEM where *=different from CDH ± SURF (p<O.OS).
•
Figure 5: pH over 8 hours
Data is presented as mean ± SEM where *=different from controis and CDH +
Ta ±
SURF Iambs (p<O.OS).
Figure 6: PaC02 over 8 hours
Data is presented as mean ± SEM where controis and CDH
+ Ta Iambs have
significantly Iower PaCa2 Ieveis from 240 minutes - 480 minutes compared with CDH ±
SURF and CDH + Ta + SURF groups (p<O.OS).
Figure 7: Pa02 over 8 hours
Data is presented as mean ± SEM. No significant differences exist between the S groups.
Figure 8: AaD02 over 8 hours
Data is presented as mean ± SEM. No significant differences exist between the S groups.
•
56
•
Figure 9: MVI over 8 hours
Data is presented as mean ± SEM where CDH±SURF were different from contraIs at 240
minutes ta 480 minutes (p<O.OS).
Figure 10: 100 x VEI over 8 hours
Data is presented as mean ± SEM where *=different from contraIs and CDH+TO Iambs
(p<O.OS).
Figure Il: Airway Pressure over 8 hours
Data is presented as mean ± SEM where bath CDH+SURF and CDH+TO+SURF groups
have Iower Paw than CDH and CDH+TO groups respectively (p<O.OS).
Figure 12: Modified Oxygenation Index over 8 hours
Data is presented as mean ± SEM. CDH is different from CDH+TO+SURF at 240
•
minutes and 360 minutes only (p<O.OS).
Figure 13: Lung Compliance over 8 hours
Data is presented as mean ± SEM where *=different from aH other groups (p<0.01).
EXPERIMENT #2:
Figure 14: Total DNA Per Lung Segment
Data is presented as mean ± SEM where *=different from PFC x 6 hrs, **=different from
operated contraIs and PFC x 12 hrs and #=different within the group (p<O.OS).
Figure 15: Rates of DNA Synthesis Per Lung Segment
Data is presented as mean ± SEM. No significant differences exist between the 4 groups.
Figure 16: Differentiai Rates of DNA Synthesis
Data is presented as mean ± SEM. No significant differences exist between the 4 groups.
•
57
Figure 1: The postulated mechanism of acute respiratory failure in CDH animalslhumans
Pulmonary hypoplasia
Pulmonary hypertension
Surfactant deficiency
Decreased pulmonary compliance
-J.-PaOz
PaCûz
-J.-pH
t
Pulmonary Arterial
Vasoconstriction
Persistent FetaI Circulation
Right -7 Left Shunting
•
Pulmonary hypertension
(Adapted from Arensman Rm and Bambini DA: Congenital diaphragmatic hemia and
eventration. In: Ashcraft KW et al. Pediatrie Surgery 3rd edition, Philadelphia, W.B.
Saunders Company, Figure 24-3, p.302, 2000)
58
•
•
•
Figure 2: Wet Lung Weight / Body Weight
3
~
l
,
*
2.5
*
.
2
~
1.5
1
Il
0.5
o
1
1
CDH
CDH+TO
CDH+SURF
CDH+TO+SURF
CONTROL
J
•
Figure 3: Dry-to.. WetLung Weight Ratios
25
ï~-"~"-"--'_·_-
"~
-------l
1
20
1
.
1
1
15
1
1
'?ft
10
1
1
i
5
!
o
1
1
-+-!-
CDH
CDH+TO
CDH+SURF
CDH+TO+SURF
CONTROL
1
•
•
Figure 4: Dry Lung Weight 1 Body Weight
•
•
•
Figure 5: pH
co
i
1
f'....
<.0
f'....
-+-CDH
--CDH+TO
-+-CONTROL
~
f'....
.
N
f'....
~CDH+surf
-.... CDH+TO+surf
f'....
co.
<.0
o
15
30
45
60
90
120 180 240 300 360 420 480
Minutes Resuscitation
•
•
Figure 6: PaC0 2
150 '
,
-+-CDH
--CDH+TO
-'-CONTROL
--*- CDH+SURF
-+- CDH+TO+SURF
100
tn
:I:
E
E
50
o
1
o
i
15
30
45
60
90
1
I
i
i
'
120 180 240 300 360 420 480
Minutes Resuscitation
•
.._ . _ - ~
Figure 7: Pa02
350
1
1
....... CDH
--CDH+TO
-'-CONTROL
--*- CDH+SURF
-.- CDH+TO+SURF
300
.
250
~ 200
ê 150
100
50
o
1
o
1
15
30
45
1
60
1
1
1
1
90 120 180 240 300 360 420 480
Minutes Resuscitation
•
•
Figure 8: Aa002
900
800
...
-+-CDH
700 -.
.-
-- CDH+TO+/-TR
-'-CONTROL
--*- CDH + SURF
-.- CDH+TO+SURF
600
500 400
f
15
i
1
30
45
60
90
120
180
240
300
Minutes Resuscitation
1
1
1
360
420
480
•
•
----l
Figure 9: MVI
600 -1
1
500
-+-CDH
--CDH+TO
....... CONTROL
-*- CDH + SURF
-+- CDH+TO+SURF
400
300
200
100
o
I
15
30
45
i
60
90
120
180
i
240
300
360
1
!
420
480
Minutes Resuscitation
----------
"---
•
1
1
Figure 10: 100 x VEI
70
T
-
i
T
1
1
60
...
50
f
j-+-CDH
--CDH +TO
-'-CONTROL
--*""" CDH + SURF
--- CDH+TO+SURF
40
30
20
110
1
1
0
*
i
15
*
j:. -nt<!/;
30
45
60
*
*
; ; ; ; ! ,
90 120 180 240 300 360 420 480
1
1
*
Minutes Resuscitation
•
•
Figure 11: Airway Pressure (Paw)
35
30
25
-+-CDH
---CDH + Ta
-ltr- CONTROL
~CDH +SURF
-0- CDH+TO+SURF
20
15
10
5
0
15
30
45
60
90 120 180 240 300 360 420 480
Minutes Resuscitation
•
•
•
Figure 12: Modified Oxygenation Index
1
140
T'--------~---------_t_
1
1
!
120
i
~
80 ~
1
1
100
1
---CDH+TO
-"lfr- CONTROL
-*- CDH+SURF
- 0 - CDH+TO+SURF
i
1
60
40 ~:::l~~~1P""-..
20
o
1
15
30
-+-CDH
1
1
1
1
1
1
45
60
90
120
180
240
Minutes Resuscitation
i
1
1
300 360 420
1
480
~
1
•
•
•
~
Figure 13: Lung Compliance
1.6
T----
1.4
J
*
*
T
1
i
~ 1.2
"""-
o
N
-+-CDH
-CDH+TO
-'-CONTROL
--*- CDH + SURF
-e-- CDH + TO + SURF
1
J:
E 0.8
o
"""- 0.6
...J
E 0.4
*
*
*
*
*
0.2
o
---
----Ji
--t-I- - , - - - - r - - - - r '- - r - - - r - - - r - I
1.5
3
4
5
6
Hours Resuscitation
7
8
1
•
•
•
Figure 14: Total DNA Per Lung Segment
9000
8000
~ 7000
.~
....... 6000
E 5000
:;( 4000
~ 3000
~ 2000
1000
~
1
#
n
tJ)
o
**
IIRPS
~LPS
-+-1-
PFC
I~-
*
x 6 hrs
PFC x 12 hrs
Operated
controls
Non-operated
controls
•
•
•
Figure 15: Rates of DNA Synthesis per Lung
Segment
0.6
0.5
-J
1
li>
«
~ 0.4
C')
IIRPS
::L
-; 0.3
~LPS
s::
~
......
~
0.2
0
0.1
0
PFC x 6 hrs
PFC x 12 hrs
Operated controls
Non-operated
controls
.
•
-
•
Figure 16: Differentiai Rates of DNA Synthesis
300
l
1
TI-~--------------------""""
1
1
250
1
1
1
•
200
~
150
100
50
o
-+-1- -
PFC x 6 hrs
PFC x 12 hrs
Operated controls
Non-operated
controls