• 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 ofExperimental Surgery McGill University, Montreal • Submitted June, 2001 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment ofthe requirements ofthe degree ofMaster ofScience

© Andreana Bütter, 2001

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Canadl TABLE OF CONTENTS Page Abstract 3 Abrégé 4 • Acknowledgements 5 Dedication 6 Abbreviations 7 Introduction 9 Review ofthe Literature: A. Normal Lung Development 11 B. Normal Diaphragm Development 12 C. Pathophysiology ofCDH 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 ofAccelerating 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 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 ofDNA 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 ofthe 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 ofGeneral 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

AaDOz = 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 ofinspired oxygen

HR = heart rate • lM = intramuscular IV = intravenous

LPS = left posterior segment ofleft 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 ofarterial carbon dioxide 7 Pa02 = partial pressure ofarterial 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 ofright upper lobe

RR = respiratory rate

RUL = right upper lobe

Sa02 = arterial oxygen saturation

SBP = systolic blood pressure

SEM = standard error ofthe 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 . Growth of both lungs is then limited by lack of intrathoracic space. At birth, the CDH possesses small, immature lungs which have difficulty adapting to extra-uterine life. Acute respiratory failure often develops with death occuring in 30-50% ofthese neonates (3). Despite many advances in neonatal care, mortality rates have remained at these e1evated levels. Prenatal ultrasound can diagnose 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 . 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 ofthis 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 ofthese 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 ofeither '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 blood­ gas 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 ofphospholipid, is not produced by type II cells but is 10cated within cell membranes. An increase in the lecithin­ sphingomyelin (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 2gth 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 ofblood gases and acidosis (Figure 1). Therapeutic interventions must target 1 or more areas in this cycle ifoutcome 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 ofpulmonary 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 ofthe compressive effect ofthe intestines on the developing lungs is the incomplete development ofthe 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 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 ofCDH 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 oftherapeutic 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 ofanimaIs are required to obtain a sufficient number ofCDH 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 ofaction 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 ofhealthy 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 (66­ 67). 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 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 (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 ofa control group makes the significance ofthese 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 4th (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 ofless 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, AaD02 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 (C02) (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 ofthe 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 ofaccelerated 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 ofthe 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 SURFACTANTATBIRTHINA SURGICALLY CREATED CDHLAMB 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 offetuses 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 ofwarmed saline through the first channel ofthe 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 ofthe 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). 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 ofthe 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 of3 kg to calculate the doses ofmedications 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 (NaHC03) 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 pre­ heparinized 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 ofIV pentothal. Simultaneously, the lamb was started on mechanical ventilation (Sechrist Infant • Ventilator Model IV-1 OOB) along with infusions of pancuronium CI mg/kg/h), ketamine 25 (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 ofthe 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 oflife 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 ifthe MAP was < 40 mm Hg or the blood pressure decreased by > 50%. Rapid visual inspection ofthe thorax for hyperinflation and lung auscultation for decreased breath sounds usually revealed the affected lung. Insertion ofa 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 ofBLES 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 ofthe 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 ofthe 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 ofthe 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 Fi02x 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 .

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• 29 EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC LIQUID INHEALTHY, 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 ofthe neck incision revealed that both the carotid and jugular veins were too small for cannulation. Our protocol was then modified to include dissection ofthe 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 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 ofthe 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 ofeach 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 ofDNA 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. Ofnote, the above analyses were performed by Dr. B. Piedboeufand 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 ofsample (RPS or LPS)

4. Rate ofDNA 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 ofDNA 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.

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• 33 RESULTS EXPERIMENT #1: FETAL TRACHEAL OCCLUSION & EXOGENOUS SURFACTANTATBIRTHINA SURGICALLYCREATED CDHLAMB 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 ofautopsy were considered as CDH ± TO ± SURF lambs. One animal was excluded because ofan 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 ofthe 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 ofthe 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 ofanimaIs 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 ofCDH ± TO ± SURF (Table 2). 34 The dry-to-wet ratios, which reflect the amount ofwater 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 ofthe 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 ofthe experiment.

•

• 36 EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC LIQUID INHEALTHY, NEONATAL PIGLETS Four groups ofpiglets were created in this set ofexperiments: (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 ofthe 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) non­ operated controls (n=4). AlI piglets were similar in terms ofheart rate, oxygen saturation, temperature, mean arterial pressure (MAP), pH, PaC02and 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 SURFACTANTATBIRTH INA SURGICALLY CREATED CDHLAMB 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 ofthe 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 oftype 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 PaC02levels for the remaining 4 hours ofthe resuscitation. In contrast, the CDH + TO group had significantly lower PaC02 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 ofdetergents 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 ofgas 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 ofprophylactic 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 extra­ corporeal 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 ofCDH.

EXPERIMENT #2: SELECTIVE PULMONARY DISTENTION USING PFC • LIQUID INHEALTHY, NEONATAL PIGLETS Turning to our second set ofexperiments involving PFC distention ofthe 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 oflung 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 ofstretch 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 • ofthese 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 ofdividing 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 ofinsufficient 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 ofRPS 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 ofthe RUL failed to accelerate postnatal lung growth. Technical difficulties and the short duration of the experiments contributed to • these negative results.

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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 plug­ unplug 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 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 ofChest Tubes

GROUP SURVIVED AGEATDEATH CHESTTUBE 8 HOURS (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 ± 0.44t 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; t = different from control (p<0.05). •

55 FIGURES Figure 1: The Pathophysiology ofCDH An illustration ofthe development ofacute 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

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

Figure 5: pH over 8 hours Data is presented as mean ± SEM where *=different from controis and CDH + Ta ± • SURF Iambs (p

significantly Iower PaCa2 Ieveis from 240 minutes - 480 minutes compared with CDH ± SURF and CDH + Ta + SURF groups (p

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

Figure 10: 100 x VEI over 8 hours Data is presented as mean ± SEM where *=different from contraIs and CDH+TO Iambs (p

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

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

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

Figure 15: Rates ofDNA Synthesis Per Lung Segment Data is presented as mean ± SEM. No significant differences exist between the 4 groups.

Figure 16: Differentiai Rates ofDNA Synthesis Data is presented as mean ± SEM. No significant differences exist between the 4 groups. • 57 Figure 1: The postulated mechanism ofacute respiratory failure in CDH animalslhumans

Pulmonary hypoplasia Pulmonary hypertension Surfactant deficiency Decreased pulmonary compliance

-J.-PaOz t PaCûz -J.-pH

Persistent FetaI Circulation Pulmonary Arterial Right -7 Left Shunting Vasoconstriction

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 1 20 1 .. 1

15 1 1 '?ft 10 1

1 5 i

! 1 1 o -+-!- 1 CDH CDH+TO CDH+SURF CDH+TO+SURF CONTROL • • •

Figure 4: Dry Lung Weight 1Body Weight • •

Figure 5: pH co i 1 f'....

<.0 f'.... -+-CDH ~ f'.... --CDH+TO -+-CONTROL N . ~CDH+surf f'.... -.... CDH+TO+surf f'.... co. <.0 o 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation • •

Figure 6: PaC02

150 ' ,

-+-CDH 100 --CDH+TO tn :I: -'-CONTROL E E --*- CDH+SURF 50 -+- CDH+TO+SURF

o 1 i 1 Iii' o 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation •

.._._-~ Figure 7: Pa02

350 1 1 300 ...... CDH .. --CDH+TO 250 -'-CONTROL ~ 200 --*- CDH+SURF -.- CDH+TO+SURF ê 150 100 50

o 1 1 1 1 1 1 1 o 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation • •

Figure 8: Aa002

900

800 ... -+-CDH 700 -. .- -- CDH+TO+/-TR -'-CONTROL 600 --*- CDH + SURF -.- CDH+TO+SURF 500 -

400 f 1 i 1 1 1 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation • •

Figure 9: MVI ----l

600 -1 1 500

400 -+-CDH --CDH+TO 300 ...... CONTROL -*- CDH + SURF 200 -+- CDH+TO+SURF 100

oIii 1 ! 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation

------"--- •

1 Figure 10: 100 x VEI

1 70 T - T i

1 1 60

... f 50 j-+-CDH 40 --CDH +TO -'-CONTROL 30 --*""" CDH + SURF 20 --- CDH+TO+SURF * 110 * * **

1 0 i j:. -nt

1 15 30 45 60 90 120 180 240 300 360 420 480

1 1 Minutes Resuscitation • •

Figure 11: Airway Pressure (Paw)

35

30

25 -+-CDH 20 ---CDH + Ta -ltr- CONTROL 15 ~CDH +SURF 10 -0- CDH+TO+SURF

5

0 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation • • •

Figure 12: Modified Oxygenation Index

------~------_t_ 1 140 T' 1 1

! 120 i 1 ~ ~ 1 100 -+-CDH 1 1

80 ~ ---CDH+TO

1 -"lfr- CONTROL 60 i -*- CDH+SURF ~:::l~~~1P""-.. 40 -0- CDH+TO+SURF

20

o 1 1 1 1 1 1 1 i 1 1 1 15 30 45 60 90 120 180 240 300 360 420 480 Minutes Resuscitation • • •

Figure 13: Lung Compliance ~

1.6 T---- * 1 * T 1.4 J

i --- 1 ~ 1.2 -+-CDH """- o 1 -CDH+TO N J: -'-CONTROL E 0.8 o --*- CDH + SURF """- 0.6 -e-- CDH + TO + SURF ...J E 0.4 * * * * * 0.2

o --t-I--,----r----r'--r---r---r-I----Ji 1.5 3 4 5 6 7 8 Hours Resuscitation • • •

Figure 14: Total DNA Per Lung Segment

~ 9000 1 8000 # ~ 7000 n * tJ) .~ ...... 6000 5000 IIRPS E ** :;( 4000 ~LPS ~ 3000 ~ 2000 1000

o -+-1­ PFC x 6 hrs PFC x 12 hrs Operated Non-operated I~- controls controls •• •

Figure 15: Rates of DNA Synthesis per Lung Segment

0.6

1 li> 0.5 -J « ~ 0.4 C') ::L IIRPS -; 0.3 ~ s:: ~LPS...... ~ 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 l 1

TI-~------"""" 300 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