POLITECNICO DI MILANO
SCUOLA DI INGEGNERIA INDUSTRIALE E DELL’INFORMAZIONE CORSO DI LAUREA MAGISTRALE IN INGEGNERIA BIOMEDICA
Anno Accademico 2017/2018
Assessment of airway smooth muscle contractility and mechanics in a model of prematurity
RELATORE: Prof. Raffaele Dellacà
TESI DI LAUREA MAGISTRALE DI:
Marco Croci 858967
Index
Sommario ... I Abstract ... VI
Introduction ... 1
Chapter 1: Respiratory System in Newborns ... 2
1.1 Neonatal Respiratory Physiology ... 2
1.1.1 Lung Development Stages ... 4
1.1.2 Airways’ Anatomy and Structure ... 10
1.1.3 Physiologic Mechanics and Regulation of the developing Airways ... 13
1.2 Neonatal Respiratory Pathophysilogy ... 15
1.2.1 Prematurity: Definition and Epidemiology ... 16
1.2.2 Prematurity: Etiology and Respiratory Pathophysiology ... 17
1.2.3 Prematurity: Mechanical Ventilation benefits and side effects ... 19
1.3 Respiratory Disorders in Preterm Infants ... 22
1.3.1 Bronchopulmonary dysplasia (BPD) ... 23
1.3.2 Bronchial Hyperresponsiveness and Asthma ... 33
1.3.3 Impact on the Health of the Respiratory System ... 38
Chapter 2: State of Art ... 41
2.1 In Vivo Studies ... 41
2.1.1 In Vivo Metodologies, Outcomes and Limits ... 43
2.2 Ex Vivo Studies ... 50
2.2.1 Ex Vivo Metodologies, Outcomes and Limits ... 51
2.2.2 Animal Models ... 61
2.3 Aims of the Study ... 64
3.1 Requirements ... 66
3.2 Bioreactor and Tissue Management Devices ... 68
3.2.1 Bioreactor ... 68
3.2.2 Tissue preparation unit ... 72
3.2.3 Microscope ... 74
3.3 Main Platform: Hardware for Signal Detection and Control ... 76
3.3.1 Power Supply Unit ... 78
3.3.2 Sensors and Signals Detection ... 79
3.3.3 Actuators and PWM Control ... 82
3.3.4 Microcontroller ... 86
3.4 Main Platform: Firmware and User Interface... 88
3.4.1 Pressure Control Modes... 89
3.4.2 Firmware Architecture ... 92
3.4.3 User Interface and Data recording ... 95
3.5 Echographic System ... 98
3.5.1 Ultrasounds physics ... 98
3.5.2 Echograph and Video Acquisition Unit ... 100
3.6 Data Processing Algorithm ... 102
3.6.1 Image Processing ... 102
3.6.2 Signal Processing ... 107
Chapter 4: Results ... 110
4.1 In Vitro Set-Up Validation ... 110
4.1.1 Evaluation of the Temperature Control ... 111
4.1.2 Evaluation of the Synchronization Unit ... 113
4.1.3 Validation of the Static Pressure Control ... 114
4.1.5 Validation of the Video Acquisition Unit ... 119
4.1.6 In Vitro test of the Signal Processing Algorithm ... 121
4.2 Ex Vivo experiments ... 123
4.2.1 Preliminary evaluation of Airway Reactivity ... 124
4.2.2 Protocol study ... 126
4.2.3 Reproducibility of the measurements ... 132
4.2.4 Airways Mechanics ... 135
4.2.5 Airways contractility ... 139
Chapter 5: Conclusions and further developments ... 148
List of Figures ... 150
List of Tables ... 153
Glossary ... 154
I
Sommario
I neonati nati prima della trentasettesima settimana di età gestazionale sono definiti pretermine; la prematurità rappresenta una delle maggiori sfide nel campo neonatale. L’aumentato tasso di sopravvivenza alla nascita ha comportato un drammatico aumento della sua incidenza e delle complicazioni ad essa correlate. Per esempio, nei soli Stati Uniti nel 2007 è stata registrata una percentuale di nascite pretermine pari al 12,7 % segnando un incremento rispetto ai due decenni precedenti vicino al 20%. In particolare, per ciò che concerne il sistema respiratorio, come evidenziato dall’ Organizzazione Mondiale della Sanità (OMS), la nascita pretermine rappresenta un evento traumatico capace di influenzare la successiva crescita e funzionalità dei polmoni gravando pesantemente sulla qualità della vita futura del bambino.
Il sistema respiratorio pretermine non raggiunge un sufficiente grado di maturità ed è dunque necessario sottoporre il bambino a ventilazione meccanica fin dalla nascita. Tali trattamenti, spesso uniti ad opportune terapie farmacologiche, sebbene siano essenziali per garantire la sopravvivenza, possono comportare una serie di effetti collaterali e causare danni importanti a polmoni particolarmente vulnerabili a stress di natura meccanica in quanto immaturi e connotati da una presenza di surfattante deficitaria per quantità e composizione.
Il surfattante è infatti un componente fondamentale nella definizione delle proprietà meccaniche e funzionali del polmone sano e la sua carenza può essere uno dei fattori che favoriscono l’insorgenza di disturbi dell’albero tracheobronchiale. Questi ultimi sono spesso correlati ad alterazioni morfologiche delle vie aeree che si traducono in limitazioni croniche della funzionalità respiratoria. Una delle principali patologie croniche è la displasia broncopolmonare, la cui incidenza nonostante le innovazioni introdotte in ambito clinico presenta tassi invariati da diversi anni. Essa determina la formazione di strutture alveolari grossolane e associate ad un insufficiente grado di ramificazione, può innescare fenomeni di fibrosi interstiziale ed influenza negativamente lo sviluppo della vascolarizzazione polmonare. Inoltre, a livello delle vie aeree inferiori la displasia broncopolmonare determina un netto aumento della resistenza al flusso d’aria e un loro marcato irrigidimento associati a una riduzione del volume corrente e ad un aumento della frequenza respiratoria. Tali modificazioni sono spesso accompagnate da infezioni e fenomeni infiammatori capaci di indurre processi d’ipertrofia della muscolatura liscia delle vie aeree. L’ispessimento del tessuto muscolare è anche favorito dall’esposizione ad alte concentrazioni di ossigeno necessarie nei primi giorni di vita per garantire una corretta ossigenazione.
Un’altra conseguenza della nascita pretermine è rappresentata dall’insorgenza dell’iperreattività bronchiale. Questo disturbo provoca un eccessivo restringimento delle vie aeree in risposta a molteplici stimoli. Essa è conseguenza di complesse modificazioni morfostrutturali associate a un
II processo irreversibile di remodeling del tessuto delle vie aeree. L’iperreattività è poi largamente studiata in quanto rappresenta una delle caratteristiche peculiari dell’asma, una malattia cronica caratterizzata dall’interazione tra processi ostruttivi ed infiammatori delle vie aeree uniti ad un insorgere del fenomeno di iperresponsività.
Gli esatti cambiamenti strutturali e i meccanismi patofisiologici che determinano un’aumentata morbidità respiratoria a seguito della nascita pretermine rimangono non pienamente compresi. Albertine et al. hanno dimostrato che agnelli meccanicamente ventilati per 21 giorni dopo la nascita presentano un ispessimento del tessuto muscolare liscio presente attorno ai bronchioli terminali rispetto agli agnelli a termine. Tuttavia, la modalità attraverso cui queste modificazioni morfologiche e limitazioni funzionali evolvono con la crescita è tuttora oggetto d’indagine.
L’obiettivo ultimo di questa tesi è il fornire nuovi strumenti investigativi per migliorare la comprensione di come la prematurità impatti la relazione tra struttura e funzione nelle vie aeree di neonati. Questo rappresenta uno studio pilota con lo scopo di implementare un set-up sperimentale, protocolli di misura e algoritmi di data processing per permettere investigazioni ex-vivo della meccanica delle vie aeree e della contrattilità della muscolatura liscia, così come l’effetto di farmaci broncocostrittori.
Nel Capitolo 1 è presentata la fisiologia del sistema respiratorio neonatale, ponendo particolare attenzione alle fasi di sviluppo polmonare a partire dalla vita fetale, all’anatomia e struttura delle vie aeree e alle loro proprietà meccaniche e funzionali. La patofisiologia respiratoria neonatale è poi illustrata partendo dalla definizione della condizione pretermine di cui sono descritte la definizione, l’epidemiologia, i principali fattori eziologici e gli effetti sulla funzionalità respiratoria tenendo conto degli eventuali disturbi e danni collaterali della ventilazione meccanica. Infine, sono introdotti i disturbi respiratori nei neonati pretermine, focalizzandosi sulla displasia broncopolmonare, sull’iperreattività bronchiale, sull’asma e sul loro impatto rispetto al sistema respiratorio.
Nel Capitolo 2 lo stato dell’arte è stato descritto secondo una classificazione che ci ha permesso di distinguere tra studi in vivo ed ex-vivo. Tra i primi sono stati investigati i risultati ottenuti in ambito clinico impiegando la spirometria, la tecnica delle oscillazioni forzate e l’endoscopia. Alla seconda categoria appartengono invece studi effettuati ricorrendo a morfometria, endoscopia, ultrasonografia e alla OCT anatomica. Per ognuna di queste metodologie e tecnologie sono stati descritti le potenzialità ed i limiti, mostrandone le peculiarità e l’efficacia nel valutare diversi aspetti legati allo stato fisiopatologico delle vie aeree. Inoltre, viene presentata una panoramica relativa all’impiego e
III alla scelta del modello animale adeguato al nostro studio. In ultimo sono discussi i principali obiettivi del nostro lavoro di ricerca.
Il Capitolo 3 è focalizzato sul sistema da noi progettato ed implementato. In particolare, dopo una breve illustrazione dei requisiti generali del set-up, viene presentata una dettagliata descrizione di ogni parte di cui esso è costituito. Quest’ultime comprendono il bioreattore, la piattaforma elettronica principale con il relativo firmware e software da noi sviluppati, il sistema ad ultrasuoni e l’algoritmo dedicato all’elaborazione dei dati.
Nello specifico il bioreattore è composto da una bagna e da una riserva, svolge le funzioni di alloggiamento e mantenimento in vita dei campioni biologici fornendo il necessario nutrimento. In questa sezione sono presentati altri dispositivi impiegati per la gestione dei tessuti: l’unità di preparazione dei campioni è usata per mantenere le vie aeree durante la fase di preparazione, mentre il microscopio è utilizzato per ottenere immagini istologiche prima e dopo i test.
La piattaforma elettronica principale è rappresentata dall’hardware dedicato alla detezione e controllo dei segnali. Tra i suoi blocchi primari vengono descritti l’unità di alimentazione, i sensori analogici e digitali, gli attuatori e la board microcontrollata. In particolare, il sistema impiega come attuatori un termometro digitale, un trasduttore di pressione analogico, due pompe centrifughe e tre termoresistenze.
Il firmware è presentato partendo da un’introduzione relativa alle modalità di controllo della pressione in anello aperto ed in anello chiuso, seguita dalla descrizione della sua architettura espressa in termini di una macchina a stati finiti. Infine, sono illustrati i dettagli relativi al software dell’interfaccia utente. La descrizione del sistema ecografico è accompagnata da un’introduzione relativa alla fisica degli ultrasuoni nella quale si è dato risalto all’impedenza ed onde acustiche ed ai fenomeni di attenuazione. Dopo questa parte sono poi riportate sia le caratteristiche generali sia le specifiche funzionalità del sistema ad ultrasuoni da noi utilizzato nel corso di questo studio.
L’ultimo paragrafo di questo capitolo è dedicato al processing dei dati e contiene la descrizione degli algoritmi di elaborazione di immagini e segnali. Nella prima sezione è illustrata la teoria relativa all’algoritmo Snake, da noi adattato per effettuare la stima della sezione trasversale interna delle vie aeree mentre nella seconda parte viene spiegato il metodo usato per calcolare le parti reale ed immaginaria dell’impedenza stimata correlando la stimolazione pressoria alla corrispettiva misura dimensionale.
I risultati sono presentati nel capitolo 4 che è organizzato in due sezioni principali relative alla validazione in vitro e alle misurazioni ex vivo. Nella prima parte sono riportati i dati relativi a diversi
IV test eseguiti sul nostro set-up al fine di valutarne la corretta funzionalità. In particolare, abbiamo testato il controllo della temperatura, l'unità di sincronizzazione, la qualità del controllo della pressione, la qualità delle immagini ultrasoniche e le performances dell'algoritmo di elaborazione del segnale. Nella seconda sezione è invece presente una descrizione completa dei risultati raggiunti, misurando il calibro delle vie aeree prima e dopo la broncocostrizione.
La prima serie di test ex-vivo da noi eseguita ha dimostrato la possibilità di caratterizzare adeguatamente le proprietà meccaniche delle vie aeree. In particolare, abbiamo utilizzato tre diverse frequenze di stimolazione (0,5 Hz, 0,250 Hz e 0,125 Hz) per sondare il comportamento e la risposta contrattile dei campioni. La frequenza più alta non ha fornito buoni risultati, principalmente a causa del basso rapporto segnale/rumore correlato alla stima dimensionale effettuata in fase di imaging. Tale evidenza suggerisce di impiegare stimoli pressori di ampiezza più elevata che tuttavia potrebbero indurre una risposta non lineare.
Inoltre, l'analisi iniziale delle proprietà meccaniche dei campioni, eseguita in assenza di un qualsiasi stimolo farmacologico, ha mostrato l’esistenza di una relazione inversamente proporzionale tra la misura del calibro delle vie aeree e il valore assoluto sia della parte reale che di quella immaginaria relative all’impedenza stimata. Tale comportamento risulta poi ancor più marcato man mano che le frequenze di stimolazione diminuiscono.
I test provocativi da noi effettuati hanno comportato l’utilizzo di farmaci quali metacolina o acetilcolina capaci di sottoporre le vie aeree a stimoli di costrizione non specifici. Questi test hanno evidenziato che il farmaco broncocostrittore induce modifiche nelle proprietà meccaniche dei campioni con un effetto tanto più elevato quanto più è il contenuto spettrale della stimolazione meccanica assume frequenze basse.
In sintesi, possiamo affermare di aver sviluppato e validato un set up sperimentale ed un protocollo di test per valutare la meccanica delle vie aeree e l'iperreattività in campioni ex-vivo.
La validazione in vitro ha fornito buoni risultati per ciò che concerne la valutazione quantitativa delle caratteristiche dimensionali delle vie aeree.
Il design di un algoritmo di controllo ad anello chiuso per il controllo della pressione ha poi permesso di raggiungere una qualità ottimale della forma d'onda di pressione, con una distorsione media da una forma d'onda sinusoidale ideale inferiore al 10%.
Per quanto riguarda la qualità delle immagini a ultrasuoni, lo studio di validazione condotto dopo un'adeguata regolazione dei parametri dell’ecografo, ha mostrato che le distorsioni introdotte sulla misura del calibro interno dei campioni sono inferiori all'1%. Inoltre, la risposta delle vie aeree alla
V stimolazione farmacologica si è protratta per l'intera durata dei test, dato da cui si evince che il bioreattore è pienamente in grado di mantenere in vita i campioni fornendo loro il corretto nutrimento. Inoltre, le indagini condotte ex-vivo hanno prodotto risultati soddisfacenti in termini di riproducibilità delle misure dimensionali e delle stime di impedenza. I risultati dei test di baseline hanno evidenziato una dipendenza dei valori d’impedenza della parete delle vie aeree dalla superfice della loro sezione trasversale. Questa dipendenza è aumentata a basse frequenze di stimolazione. I test farmacologici hanno mostrato risultati coerenti con le diverse caratteristiche delle vie aeree studiate. Pertanto, il nostro dispositivo e i nostri protocolli sono stati in grado di fornire misure affidabili e sensibili ai cambiamenti indotti dalla broncocostrizione.
La tecnologia che abbiamo sviluppato può dunque essere utilizzata in studi futuri per caratterizzare la meccanica delle vie respiratorie degli agnelli, i quali possono altresì presentare differenti livelli di sviluppo respiratorio ed esser sottoposti a molteplici trattamenti terapeutici dopo la nascita, come periodi di ventilazione meccanica (da tre giorni a 21 giorni) o interventi di varia tipologia (ad esempio invasivi o non invasivi). In ultima analisi, tale tecnologia permette di indagare come le diverse strategie di ventilazione e i necessari trattamenti di supporto alla vita influenzino i fattori che abbiamo esaminato, fornendo una conoscenza che potrebbe aiutare nello sviluppo di protocolli clinici migliorati e volti a ridurre sia l'incidenza che la gravità delle complicazioni respiratorie a lungo termine della prematurità.
VI
Abstract
Infants born before 37th week of gestational age are defined preterm. Prematurity represents one of the major challenges in the neonatal field. The increased survival rate at birth has led to dramatic rise in its occurrence and of the complications relative to it. For example, in 2007 the number of preterm delivery has been registered as around 12.7% in the United States, reporting an increment of approximately 20% with respect to the two precedent decades. In particular, for what concerns the respiratory system, as underlined by the World Health Organization, preterm delivery represents a traumatic event, able to influence lungs’ subsequent growth and functionality, deeply affecting the quality of life of the newborn.
Since the respiratory system has not reached a sufficient stage of maturation, it is often necessary to help the newborn with ventilatory supports from birth. Although such treatments, often joined with proper pharmacological therapies, are essential in guaranteeing the survival of the patient, they may lead to a series of side effects and cause relevant impairments to immature, surfactant-deficient lungs. Surfactant is indeed a fundamental component in the definition of the mechanical and functional properties of the healthy lung, and its shortage could be one of the factors that facilitate the insurgence of disturbances of the tracheobronchial tree. These are often correlated to alterations in the morphological structure of the airways and frequently translate in chronic limitations of the respiratory functionality.
One of the principal chronic pathologies belonging to this category is the Bronchopulmonary dysplasia which, despite all the new innovations introduced in the clinical field, presents rates of incidence unchanged throughout several years. The consequences of this disease include the growth of simplified and larger alveolar structures with a decreased level of alveolarisation, increased interstitial fibrosis and abnormal pulmonary vasculature. Furthermore, at the lower airway level, this disease causes a sharp increase in the airways resistance as well as a decrease of their compliance, leading to a reduced tidal volume and an increased respiratory rate. These modifications are often accompanied by infection and inflammatory phenomena, which may lead to airway smooth muscle hypertrophy. The thickening of the muscular tissue is also correlated to a high exposition to large quantity of oxygen in the first days of life, which is necessary to guarantee a correct oxygenation. Another consequence of the preterm birth is the bronchial hyperresponsiveness, a condition characterized by an excessive airway narrowing in response to a variety of stimuli that do not cause any response in healthy subjects. It is a consequence of an ongoing and irreversible process of morpho-structural remodeling of the airway wall tissue. The bronchial hyperresponsiveness is widely studied as it is one of the most dangerous features of asthma, a chronic disease characterized by an interplay between airflow obstruction, hyperresponsiveness and airways inflammation.
VII The exact structural changes and pathophysiological mechanisms of increased respiratory illness following preterm birth remains not fully understood. Albertine et al. demonstrated that preterm lambs mechanically ventilated from birth for 21 days had thickened smooth muscle area around terminal bronchioles compared to term newborn lambs4. However, how these morphological changes and functional impairment evolve with growth is still unknown.
The aim of this thesis is to provide new investigational tools for improving our understanding on how prematurity impacts the relationship between structure and function in the newborn’s airways. This is a pilot study with the purpose of implementing an experimental set-up, measurement protocols and data processing algorithms for allowing the ex-vivo investigation of airways mechanics and airway smooth muscle contractility as well as the effect of bronchoconstrictor drugs.
Our thesis is organized in five chapters:
Chapter 1 presents the physiology of the neonatal respiratory system, giving particular relevance to the lung development stages during fetal life, to airways anatomy and structure and to the physiology, mechanics and regulation of developing airways. The neonatal respiratory pathophysiology is also described by illustrating definition and epidemiology of prematurity, as well as its etiology, respiratory pathophysiology and respiratory supports benefits and side effects. Respiratory disorders in preterm infants are presented with a particular focus on bronchopulmonary dysplasia, bronchial hyperresponsiveness and asthma, as well as their long-term impact on the respiratory system.
In Chapter 2 the state of art is exhibited according to a classification between in vivo and ex vivo studies. Among the first category the clinical results related to spirometry, forced oscillation technique and endoscopy, whereas in the second one morphometry, endoscopy, ultrasounds and anatomical optical coherence tomography have been illustrated as techniques able to effectively study ex vivo samples. For each of these two areas the methodologies, outcomes and limits have been evaluated, as well as their peculiarities and effectiveness in evaluating different respiratory aspects associated to the pathophysiological status of the airways. Furthermore, the purpose and the choice of the appropriate animal model to be used for our study is discussed. Finally, the aims of this thesis are presented.
Chapter 3 focuses on the system we have designed and developed. The chapter starts with an illustration of its general requirements, followed by the detailed description of every component. In particular, these consist in the bioreactor, the main electronic unit with its relative firmware and software, the ultrasound system and the algorithms dedicated to the data processing.
VIII The bioreactor is composed by the tissue bath and the reservoir; it is dedicated to the housing of the samples and it keeps them alive and provided with proper nourishment. In this section other tissue management devices are also presented: the tissue preparation unit, which is used to maintain the samples during the preparation phase, and the optical microscope utilized to obtain histological images both before and after the tests.
The main electronic platform is dedicated to the signal detection and control. Its main blocks are the power supply unit, analog and digital sensors, actuators and the microcontroller. In particular the system employs one digital temperature and one analogic pressure sensor, whereas two pumps and three thermo-resistances as actuators.
The firmware is described beginning with an introduction related to the pressure control modes, which presents the open loop and closed loop strategies; then the firmware architecture is illustrated, with a particular focus on the finite state machine developed, followed by a description of the user interface software.
The ultrasound system is presented with a brief introduction related to the ultrasound physics in which the main focus is related to the acoustic waves, acoustic impedance and attenuation. After this, the ultrasound system used in this study is described.
The last section of this chapter is dedicated to the data processing, which features the image processing and the overall signal processing algorithms. In the first section it is described the theory behind the Snake algorithm, which is the algorithm chosen for the measurement of the cross-section of the biological samples. In the second section the computation algorithms for the estimation of the real and imaginary part of the impedance between the pressure inside the airway and its dimensional characteristics are presented.
The results are presented in Chapter 4, which is organized in two main sections, the in vitro validation and the ex vivo measurements. In the first part several evaluations performed on our set-up in order to test its proper functionality are presented. In particular we tested the temperature control, the synchronization unit, the quality of the pressure control, the quality of the ultrasonic images and the capabilities of the signal processing algorithm. In the second section there is a complete description of the results achieved, measuring airway mechanical behavior before and after bronchoconstriction. The first ex-vivo set of tests demonstrated the possibility to adequately characterize airways mechanical properties. In particular, we used three different stimulation frequencies to probe the samples (0.5 Hz, 0.250 Hz and 0.125 Hz). The highest frequency did not provide good results, mostly because of low signal-to-noise ratio on the imaging signals, suggesting the necessity of higher amplitude pressure stimuli which, however, might induce non-linear response. However, as this
IX frequency is far from the physiological rhythm of the spontaneous breath, this issue simply suggests focusing the study on lower frequencies.
In addition, the initial analysis of the airways mechanical properties performed through the impedance estimation, before the pharmacological stimulus, presented an inversely proportional relation between the dimensional measurement and the absolute value of both the real and imaginary part. This behavior is increased with decreasing stimulation frequencies.
We also performed bronchoconstriction tests using methacholine and acetylcholine to study the airway response to non-specific constriction stimuli. These tests highlighted that the bronchoconstrictor drug causes modifications in the mechanical properties of the samples, and its effect is higher at lower frequencies.
In conclusion, we developed and validated an experimental set up and test protocols for assessing airway mechanics and hyperreactivity in ex-vivo samples.
The in vitro validation provided good results for the quantitative assessment of the AWs geometry. The design of a closed loop control algorithm for pressure control allowed reaching an optimal quality of the pressure waveform, with an average distortion from an ideal sinusoidal waveform lower than 10%.
Regarding the ultrasound image quality, after appropriate tuning of the ultrasound scanner parameters, the validation study performed showed that geometrical distortions are lower than 1%. Moreover, the samples responded well to drug stimulation for the whole duration of the tests, allowing us to state that the bioreactor is fully able to keep specimens alive furnishing their cells with the correct nourishment.
The ex-vivo investigations performed yielded satisfactory results on the reproducibility of the dimensional measurements and on impedance values. Baseline measurement of mechanical properties of the airways showed a dependence of airway wall impedance on internal area. This dependence is increased at low stimulation frequencies. Test with bronchoconstrictors showed results coherent with the different characteristic of the airways studied. Therefore, our device and protocols were able to provide reliable measurements, sensitive to changes induced by bronchoconstriction.
The technology we developed can be used in future studies to characterize airways mechanics of lambs with various respiratory developmental stages and subjected to different respiratory treatments after birth, such as different mechanical ventilation periods (three days to 21 days) or techniques (i.e. invasive or not invasive). Hence, the investigation of how the ventilation strategies and supports at birth do influence the factors we examined could be deepened, and, therefore, this knowledge might
X help in designing improved clinical protocols aimed to reduce both incidence and severity of long term respiratory outcomes of prematurity.
1
Introduction
Preterm birth is a severe impairment for the future health of the respiratory system. More than 10% of live births occur before the completion of 37 weeks of gestation in the United States and Europe. Prematurity is associated with serious respiratory illnesses that are especially problematic in the first two years of life with frequent need of primary care visits and re-hospitalization because of respiratory tract infections and airway hyper-reactivity1. While a diagnosis of bronchopulmonary dysplasia (BPD) is associated with a higher frequency and duration of these symptoms, it does not predict whether a preterm infant will experience long-term complications. Indeed, these symptoms are often overcome with age, however they do cause numerous problems and impairment to children. Similarly, preterm infants without a diagnosis of chronic lung disease may experience respiratory morbidities early in life and present with respiratory limitations at school age and into adulthood1–3. The exact structural changes and pathophysiological mechanisms of increased respiratory illness following preterm birth remains not fully understood. Albertine et al. demonstrated that preterm lambs mechanically ventilated from birth for 21 days had thickened smooth muscle area around terminal bronchioles compared to term newborn lambs4. However, how these morphological changes and functional impairment evolve with growth is still unknown.
The final aim of this thesis is to provide new investigational tools for improving our understanding on how prematurity impacts the relationship between structure and function in the newborn’s airways. This is a pilot study with the purpose of implementing an experimental set-up, measurement protocols and data processing algorithms for allowing the ex-vivo investigation of airways mechanics and airway smooth muscle contractility as well as the effect of bronchoconstrictor drugs. The main focus is to evaluate the relationship between the morphology of the airway and the functional parameters, namely finding how the anatomical changes correlate with functional impairment.
These studies have been performed by means of a specifically designed and developed device used on tissue samples provided by the animal model developed at the Kurt Albertine laboratory at the University of Utah in Salt Lake City. This gave us the unique opportunity to test and study biological samples immediately after the euthanasia of the animals, which could have been treated in several different ways, such as different mechanical ventilation periods (three days to 21 days) or techniques (i.e. invasive or not invasive) to mimic typical pretem babies clinical history.
2
Chapter 1: Respiratory System in Newborns
1.1 Neonatal Respiratory PhysiologyRespiration is a complex phenomenon which involves different structures able to grant both the oxygen provision and the carbon dioxide removal respectively from the external environment and blood, achieving the gas exchange process necessary to support life (see
Fig.1.1)5. In particular, the respiratory system is formed by the upper and lower airways (AW). The nasal cavity, sinuses, nasopharynx, and the prossimal tract of the larynx, belong to the former and are mainly deputed to filter and warm the air recruited through inspiration. Below the vocal cords, the latter includes the distal tract of the larynx, trachea, bronchi, and lungs which enclose and protect, by means of the pleuras, bronchioles, alveolar ducts and alveoli.
Pulmonary ventilation, constituted by the alternate phases of inspiration and expiration, is presided by the respiratory muscles among which the most important are the diaphragm, and intercostal muscles. Moreover, blood perfusion of the lungs is ensured by the pulmonary circulation: on one hand the carbon dioxide-full blood (PO2=40mmHg; PCO2=45mmHg) is carried, by pulmonary arteries and blood vessels from the heart to the lungs, on the other the oxygen-full blood (PO2=105mmHg; PCO2=40mmHg) passes through the opposite pathway by means of pulmonary veins and blood vessels. Obviously, a proper development of the above mentioned components is necessary to guarantee the newborn’s survival and the normal metabolism at birth. Moreover, the process of lung growth during fetal life is related to 5 distinct phases6. Each of them occurs during a precise period of gestational age (GA) and presides over specific organogenesis mechanisms, capable of determining the morphostructural and histological changes necessary for the maturation of the respiratory system. Specifically, these phases, which would be described in detail in the next paragraph, include: embryonic, pseudoglandular, canalicular, saccular, and alveolar stages (see Tab.1.1)7.
3
4
1.1.1 Lung Development Stages Embryonic Stage (3 to 7 weeks’ GA)
About on GA 22th day in an outgrowth of the primitive foregut endoderm called medial pharyngeal groove, a wideninig of the posterior part of the laryngotracheal sulcus forms the structure from which the lung will take its origin. Just few days later the embryo reaches 3mm of length and the lung bud begins its development parallely and close to the primitive esophagus. Furthermore, the primitive respiratory epithelium covers the splanchnic mesenchyme, starting the process that lead to the bronchial tubules’ formation. Later the latter will branch repeatedly constituting the upper part of the tracheobronchial tree8.
The primary bronchial structures, which presides the development of the main stem, appear on GA 28th day from the lung bud’s bifurcation while the upper tract will become the larynx and the trachea (see Fig.1.2 A-D). The fifth week of GA (see Fig.1.2 E-F) underlines the beginning of a second process of branching in which the right and left pulmonary lobes develop asymmetrically. During the subsequent week it is possible to observe, after another turn of branching, 10 and 9 segmental bronchi respectively in the right and left lung. They will represent the bronchopulmonary segments of the mature lung (see Fig.1.2 G-H)9.
Along this initial maturational phase, the lower AW are not formed at all yet. Indeed, the cell populations that constitues the mesenchyme are still primitive, and the differentiation mechanism that will lead to the appereance of the airways smooth muscle (ASM), and other connective components, such as cartilage, has not started.
However, the esophagus and trachea are well separated and clearly detectable at the end of the embryonic stage, while the autonomic innervation of the lungs takes place. In particular the ganglion cells reach the tissues that surround the trachea by the GA 50th day and form the neural plexus whose nerve fibers will extend up to the bronchi.
Finally, the pulmonary vascular plexus connects with the heart and, for the whole length of the fetal life, the right ventricle ejects its blood bypassing the pulmonary vascular bed by means of the vascular connection between aortic arch and the pulmonary artery10.
5 Fig. 1.2: Schematic representation of lung development during the first 7 GA weeks.
6
PseudoGlandular Stage (5 to 17 weeks’ GA)
The gradual maturation of the lung’s bronchial district (see Fig.1.3) signs the pseudoglandular phase of fetal respiratory development. Specifically, during the branching morphogenesis process the segmental tubules of the still immature lungs are subjected to a repetitive dichotomous branching which leads to the initial stage of bronchial tree formation.
By the seventeenth week of GA, the conducting AWs have almost reached their complete stage of development. The terminal bronchioles are well discernible, while in the upper, middle and lower lung lobes it is possible to find up to 17,23 and 23 generations of bronchial tubules respectively11. Besides, in the peripherical part of the lungs the distal branches of the bronchioles give arise to acinar buds that will become an essential component of the mature pulmonary acinus.
Along this stage, the pseudostratified epithelium undergoes some structural modifications related to the branching process: in upper AW a tall columnar epithelium is formed, while in the acinar tubules it assumes a cuboidal shape.
Moreover the fetal ASM, whose contractility had been observed in numerous ex-vivo studies, and cartilage extend as far as the respiratory bronchioles and the segmental bronchi. The active mucus productions starts by the GA 100th day9. Indeed, at about 8 weeks’ GA, a complex network of neural fibers innervates the envelope of the AW and the layer of ASM. The pseudoglandular phase marks also some important steps of pulmonary vascular system’s development.
In particular, on one hand the angiogenisis process leads to the pulmonary arteries formation, which occurs parallely to the bronchial tubules, and on the other it presides the lymphatic vessels and pulmonary veins growth through the interlobular connective tissue septa. Finally, the vasculogenesis process allows the widespread perfusion of the peripheric lung parenchyma, leading to the preacinar blood vessels creation10.
7
Canalicular Stage (16 to 26 weeks’ GA)
During the canalicular stage, the multiplication of vascular capillaries in the lung interstitium has a fundamental role in the alveolar-capillary membrane development without which the correct functionality of the pulmonary gas-exchange surface could not be assured (see Fig.1.4).
The blood-air barrier formation, the synthesis and subsequently secretion of the surfactant fluid represent a sensitive and critical point of the fetal respiratory life. The related eventual problems or malformations could seriously endanger the survival of the newborn infant after the delivery and could lead to hypoxemia, a condition that is not compatible with life after birth. Indeed the respiration cannot occur unless the vascular canals are sufficiently close to the alveolar tissue, in order to allow oxygen and carbon dioxyde diffusion.
The active surface of the alveolar-capillary membrane raises exponentially its extension determining in the meanwhile a diminuition of the mesenchymal wall thickness6. Besides, already in the initial days of this stage the tracheobronchial tree is complete and the terminal bronchioles begin to split themselves into two or more respiratory structures which will form groups of small acinar tubules enveloped by cuboidal epithelium.
Later the mature pulmonary acinus will arise from these buds which will be subjected to a differentiation phenomenon able to form the adult respiratory unit. The latter is characterized by the presence of some bronchioles whose distal part will branch in 6 to 7 generations of alveolar ducts and alveoli.
Moreover, the angioblastic cells that populate the mesenchyme preside the increase of the interacinar capillaries’s number which are aligned close to the air spaces in order to grant the optimal connection to the adjacent epithelial tissues belonging to the blood-air barrier10.
Finally, from an histological point of view, at the end of GA 26th week it is possible to detect well differentiated mucus and ciliated cells belonging to the conducting AW, while cartilage, submucosal glands, and ASM present the same morpho-structural distribution and conformation observable in the mature lung9.
8
Saccular Stage (24 to 38 weeks’ GA)
The saccular stage of the fetal lung growth is marked by the dilatation and expansion of the terminal groups of acinar tubules which lead to the thin transitory alveolar ducts and saccules’ formation (see Fig.1.5).
This process is accompanied by a drastic reduction or compacting of the surrounding mesenchymal tissue. At the same time the lung keeps on its development supporting the peripheral branching and the growth of three additional generations of transitory alveolar ducts. The latter end in the primary saccules which give name to this phase6.
Moreover, the amount of delicate collagen fibers increases in the intersaccular and interductal septa which also contain a double capillary network, while the epithelium proliferation slows as result of an almost complete process of cytodifferentiation.
Despite this fact, the concentration of pulmonary surfactant is still low and its biochemical composition is deeply different if compared with those that are present in the health newborn and the basal lamina of the endothelium and epithelium haven’t completely formed the thin-walled blood-air membrane yet12. After the GA 36th week, the stroma, which constitues the lung interstitial tissue, raises both its extra-cellular matrix and elastine amounts. In particular, elastine is located in regions that will be the future housing of the interalveolar septa which in turn will split the terminal alveolar saccules into true alveoli. At the end, from a biomechanical point of view, the lungs present only little elastic recoil and could be heavily damaged by any sort of barotrauma6.
9
Alveolar Stage (36 weeks’ GA to 2 years’ Postnatal)
The final phase of the respiratory system’s development is the alveolar stage (see Fig.1.6).
It presides the growth of secondary alveolar septa that are responsible of the transitory ducts and terminal saccules’ subdivision into true alveolar ducts and alveoli6.
Besides, the alveolar-capillary membrane completes its maturational development making available a broad and active surface for gas exchange to the lungs.
Finally, the remodeling of the pulmonary circulation and capillary bed results in a diminution of the pulmonary vascular resistance, while a process of rapid cellular proliferation in both the epithelial and mesenchymal cell populations occurs10. The latter determines the formation of a tight epithelial barrier which extracellular fluid and ions are not able to overcome by diffusion.
However, in the first months of life the lungs remain very vulnerable and could be easily injured by infections or mechanical stresses.
10
1.1.2 Airways’ Anatomy and Structure
Although several models have been proposed, the implementation of a complete, simplified and universally accepted model of the human tracheobronchial tree is still a complex challenge. The most widespread and recognized in the scientific community is the Weibel’s model, which assigns a number to the AW generations, starting from the trachea (gen. 0) and ending with the alveolar sacs, that have the highest number (gen. 23)13.
In particular Weibel’s approach hypothesizes that each AW split itself into two deeper structures characterized by equal dimension and similar properties assuming that the number of branches belonging to gen.(n) is half that in gen.(n+1).
This approximation implies that the number of air passages belonging to the gen.(n) is given by the following exponential relation: 2nd . For example, the segmental bronchi are classified as gen.(4) and their number is actually equal to sixteen. However the idea that the AWs develop following a perfect dichotomous pattern is contradicted by the evidence: the peripheral AWs of the same generation often have different length and size and the presence of trifurcations is quite common.
In the past years, the use of imaging techniques based on computer tomography allowed the three-dimensional rendering of the AWs’ structures (see Fig.1.7) and proved the validity of Weibel’s model until the sixth generation. This investigation also demostrated that some AWs reach their end at the level of gen.(8) or give rise each one to three or more branches14.
11 Anatomically speaking, the three main components of the mature AW are: epithelium, cartilage, and smooth muscle (see Tab.1.2)15.
Along their whole length the AWs are lined by the epithelial cells which belong to eight different cell types. Among these the most important is the columnar epithelial cell, which presents a layer of cilia on its apical surface, while the other cell types include basal cells, brush cells, small granule cells and mucus-secreting cells.
Although the cartilagineous tissue is present from the trachea to the bronchioles, it changes profoundly its morphological structure and mechanical properties as soon as the AW generations become deeper and deeper. In the trachea it forms C-shaped rings that are open posteriorly while in the bronchi the cartilage appears as plates which substain the whole AW lumen. Besides, in the lower, distal AW generations these plates become smaller and heterogeneously distributed, progressively disappearing near the bronchioles’ level.
In the trachea, the ASM is limited to the trachealis muscle. Instead in the bronchi it creates a complete circumferential envelope that becomes thinner and more discontinuous along the lower generations. The inner trachea’s surface is covered and protected by the mucosal and submucosal layers. The latter is constituted by connective tissue that merges to the cartilage rings’ perichondrium and hosts the blood, lymphatics vessels and mucus-secreting glands. Finally, the adventitia surrounds the external part of the trachea and connects it to some adjacent structures such as the neck musculature, and the esophagus.
The principal innervation of the tracheobronchial tree’s upper tract derives directly from the vagus nerve16. This neural plexus presides the cholinergic stimulation which modulates the ASM contractility, while other neural fibers control bronchial reactivity through a nonadrenergic stimulation mediated by neuroactive substances, like nitric oxide. The circulating catecholamines provide also a sympathetic control of the AW that produces very bland effects on the respiratory metabolism.
12 Tab. 1.2: Structural characteristics of AWs.
13
1.1.3 Physiologic Mechanics and Regulation of the developing Airways
The main contribution of the AWs to respiratory mechanics is undoubtedly resistive. Indeed, they modulate the resistance to air-flow, whose main part, both in healthy adults and infants, is determined by the upper and larger AWs. Moreover, the peripheral branches’ cross-sectional surface raises, reaching very high values (see Fig.1.8 A) after the gen.(8) and minimizing the opposition to air passage (see Fig.1.8 B).
The latter becomes very difficult to study within the lungs, especially if compared to the theoretical gas-flow which passes through ideal tubes, and is characterized by both laminar and turbolent components.
Regarding the AWs’ compliance, the Literature agrees on the fact that the newborn infant's AWs are more compliant than those of the adult (see Fig.1.9)17. This means that the immature AWs are more easily deformed and damaged by a barotrauma than the stiffer AWs of the older child, which also present different mechanical properties. These properties and the mechanisms at the base of developmental changes in AWs function had been widely studied using animal tracheas as models. For example, an in vivo investigation about the perfused and innervated lamb’s trachea proved an age-dependent diminution not only in AW compliance but also in the trachea’s time constant of relaxation18.Besides, it was demonstrated that newborn lambs’ pressure-flow characteristic of the trachea is influenced by the ASM tone. Indeed, the layer of ASM is functional already in the first GA trimester and it modulates the AW’s phasic spontaneous events of relaxation and narrowing, which are accompanied by back-and-forth movement of lung fluid. The ASM at 23 weeks’ GA is present in all the generations that belong to the conducting AW, as evidenced by human autopsy data, and could also provide an essential support to the lung growth by supplying an intraluminal positive pressure19.
14 Generally speaking, the pharmacological stimulation triggered by bronchoconstrictor drugs’ (e.g. acetylcholine) increase the AW’s stiffness, making them less deformable and more resistant to airflow by means of their lumen narrowing. Moreover, the Literature suggests that the changes in AW’s mechanical properties induced by these pharmacological agents are age-dependent and influenced by the lung developmental stage.
However, the way in which the ASM’s maximal tension development and sensitivity to different agonist drugs are affected by postnatal growth is still not well known20. On one hand some investigators claim that sensitivity and contractility rise with age, while on the other some studies assert that these two properties achieve their peak in the period immediately subsequent to birth, declining later. In the lambs instead, there is a widespread agreement that active stress reaches significant and increased maximum values in the final part of the fetal life. Definitely, the role played by the respiratory maturation process on AW’s contractile response remains not fully explained, although some in vitro physiologic studies ascerteined a reduced non-adrenergic responsiveness in early postnatal life employing isolated tracheal ASM strips from different species16. The protocols followed during these investigations had also assured a careful normalization of the ASM’s contractility with respect to myosin content and tissue mass. In conclusion, despite a clear evidence of the presence of physiological maturational changes in AW’s function that should still be adequately studied, it is also important to focus and underline the effects on AW’s reactivity related to compromised physiopathological conditions, whose details will be discussed in the next paragraphs.
15
1.2 Neonatal Respiratory Pathophysilogy
Introducing the neonatal respiratory pathophysiology, one should remember that several different AWs’ abnormalities and malformations, which could lead to serious chronic diseases, have origin during both fetal and post-natal stages of lung development.
They are also often related to the preterm condition, which represents per se a high risk for infants’ survival. Indeed, it is estimated that about the 30% of the 4 million annual neonatal deaths in the world is strictly correlated to prematurity and the proportion of infantile deaths due to it varies among countries (see Fig.1.10)21.
For this reason, in the past years, the integration between investigations performed in vivo on human patients and more invasive studies carried out using several animal models had resulted in a considerable effort aimed to increase the knowledge about the respiratory function impairment in the preterm newborns.
In particular, a better understanding of the underlying mechanisms related to AWs and lungs pathologies is an essential premise in order to achieve important improvements in pediatric health care domain.
The next paragraphs focus on prematurity definition and peculiarities and present an overview of its outcomes on the respiratory system, taking in account the side effects due to necessary support-life treatments.
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1.2.1 Prematurity: Definition and Epidemiology
According to the medical Literature, we can speak of preterm birth (PTB) when the delivery occurs between the 20th and the37th week of GA. The degree of newborn’s prematurity is also classified by GA as extremely preterm, very preterm or moderate preterm depending on the fact that the child is born before 28,32 or 37 weeks of GA respectively.
An accurate categorization of prematurity by tipology and GA is fundamental for assuring a rigorous trends’ monitoring and an accurate estimate of health service needs. Clinical data gathered along the past two decades show a dramatic increase especially in near term PTBs’rates. In 2007, the U.S. PTB rate was 12.7%, determining a raise of about 20% in less than twenty years22.
In particular, the frequent use of assisted reproduction techniques or new obstetric surgery trends related to changes in clinical practice (e.g. surgeons are now more inclined to perform Cesarean sections) and the rising number both of expectant women over 34 years of age and of multiple births, contributed in determining this trend.
Moreover the introduction of ultrasound techniques in order to assess GA might have led to a larger number of PTB diagnosis, while inconstant and erroneous classifications of fetal losses, spontaneous abortions and early neonatal deaths as PTB could have also concurred to the worrying raises of PTB rates23.
Despite eventual recording and diagnostic errors, prematurity represents a major challenge for neonatal care and is one of the most important causes of infantile mortality and morbidity.
Besides, on one hand the global incidence of prematurity is just less than 10% and it means that over 12.5 million children are born preterm every year, but on the other the larger part of PTBs (85%) occurs in Africa and Asia.
Indeed, in many under-developed countries, whose neonatal mortality rate is often higher than 45 per 1000 live births, the most part of neonatal deaths has some preventable complications such as asphyxia and infections and not the PTB as main cause.
Conversely, in the United States and in E.U. countries, whose neonatal mortality rate is less than 15 per 1000 live births the fraction of deaths due to PTB increases over the 35%, since other potential risk factors are usually solved and properly treated21.
These marked inequalities in the etiological distribution of infantile deaths are also an evident indicator of dramatic health care differences between low and high resource settings.
Briefly, the number of preterm children seems to be raising worldwide and in relation to a heterogenous situation, characterized by deep differences often within the same country.
17
1.2.2 Prematurity: Etiology and Respiratory Pathophysiology
From a patophysiological point of view, prematurity, whose etiology is correlated to different factors and is not utterly explained, is a complex condition which involves multiple systems and organs, influencing both their development and function.
In the most part of spontaneous PTBs, causal mechanisms which lead to a preterm delivery are related to maternal stress, an exaggereted inflammatory response, a decidual hemorrhage or myometrial stretch. Each of these main causes activates a precise pathway able to determine an increase in the level of prostaglandis and proteases triggering the labor by means off uterine contractions.
Moreover, other risk factors for PTB include genetic and biological predispositions, fetal diseases or characteristics, enviromental influences, and also a compromised maternal psychological, nutritional or health status24.
On one hand some studies underline also the important role of gene–environmental and gene-gene interactions, which could account for the ethnic disparities in the PTB incidence while on the other there is no doubt that the access to adequate health care services is able to minimize these risk factors. For example, the use of corticosteroids before birth or during preterm delivery has been shown to be an efficient and a cost-effective help in supporting the pulmonary development of the infant and in minimizing the issues due to respiratory distress syndrome which represent the first cause of disability and death in extreme preterm newborns25.
Indeed, according to the World Health Organization (WHO), PTB represents a traumatic event for the respiratory system, which influence the lungs’ subsequent growth and functionality and could deeply affect the child’s future quality of life.
The WHO also stresses the importance of an accurate monitoring and treatment of respiratory conditions during the first days of preterm newborn’s life in order to avoid serious injuries and impairments22.
In particular in the U.S., as recorded by the “Centers for Disease Control and Prevention”, while a diagnosis of asthma is formulated for about the 10% of the whole infantile population, this percentage is three times higher among the children who experienced prematurity condition (see Fig.1.11)26. This data has substantially been confirmed by several studies which, accessing large clinical datasets, have found an elevated asthma risk in preterm children compared with the healthy control groups. The coefficient of asthma risk seems also to increase in a directly proportional way to the prematurity’s degree. The investigations performed by Hack et al., for example, had provided longitudinal data which report that almost one of four among extremely preterm children would be treated for asthma’s symptoms at 8 years of age27.
18 In conclusion, the recognition of PTB as an intrinsically dangerous condition for children’s health and respiratory development, also in the case of moderate prematurity, should induce the physicians to accurately assess the patient for eventual signs of AW disease in the early period of necessary hospitalization.
Further studies of pulmonary pathophysiology and clinical data collected from extremely preterm babies or through animal models, will hopefully let to optimize the therapeutic choices and long term treatments of this high-risk population.
19
1.2.3 Prematurity: Mechanical Ventilation benefits and side effects
The high rate of preterm newborns’ survival, reached in the past decades, would not have been possible without the introduction of mechanical ventilation (MV) in the neonatal intensive care units. The use of positive-pressure mechanical ventilators, which replaced the negative-pressure “iron lungs” during the ‘50s, had drastically improved the therapeutic outcomes of respiratory treatements both in adults and infants. The MV represents an irreplaceable life-support instrument and could be controlled either in pressure or flow and evaluated in terms of duration and modality.
Since the largest part of preterm infants has to be ventilated after PTB for a period of time of variable lenght depending on patophysiological condition’s severity, the MV and prematurity are intrinsicly correlated and this interdependence should be taken in account before performing any sort of respiratory analysis on preterm babies.
If on one hand MV provides an essential contribution in the mortality rate’s decrease among children who experienced a PTB, on the other its side effects have a leading role in the morbidity rate’s increase since the pressures erogated by MV and O2 inspired fraction are often extreme for an immature and surfactant-deficient lung (see Fig.1.12)28.
20 In particular a lung characterized by a low
compliance is more vulnerable to ventilator induced injuries which can lead to a serious respiratory dysfunction. These injuries include pulmonary edema, progressive respiratory failure and atelectasis.
Besides, while in the most part of clinical cases MV has a negligible effect on mature AWs, it is able to modify the morphological structure and mechanical functionality of preterm infant’s ones. The severity of these changes in terms of deformation, which seems to be inversely related to age, increases when the AWs’ stiffness is abnormally low. For example, investigations performed using preterm lamb as animal model have observed a raise in tracheal lumen, lesions of the epithelial layer and a diminution of ASM thickness. According to
Fig.1.13, after MV treatment (group II) the trachea
shows a raised collapsibility and requires a higher respiratory work for its expansion in comparison to the control group (group I)29.
The effects of MV on lower AWs generations are still not well known. Some qualitative dimensional measures have suggested that the contractility of ASM and the presence of regional differences in tissue’s histologic composition and conformation might exasperate the effects of MV on the peripheral portion of pulmonary parenchyma.
The complications induced by MV can also lead, if the period of assisted respiration persists for several days, to the development of gross and enlarged alveolar units. These structures determine the narrowing of distal AWs’ diameter and consequently the increase of their baseline resistance to the air-flow.
Moreover, if the MV is accompanied by a prolonged hyperoxic exposure, the tethering of intraparenchymal AWs results less compact and also the number of bronchiolar-alveolar attachments is reduced30.
21 Indeed, although the exposure of the preterm infants to an increased oxygen fraction is a widespread clinical practice in order to ensure the correct O2 blood saturation, it may also cause some dangerous side effects. For example, it has been proved that in guinea pig pups a supplemental rate of oxygen is associated to an incremented ASM surface and, even if there are not clear evidences (recent studies demonstrate that 40% O2 inspired fraction determined a greater raise in AW reactivity than 70% O2 exposure, which instead elicited a greater ASM thickening), it seems probable that this modification may be related to the onset of AWs hyperreactivity (BHR)31.
However, BHR is a complex phenomen which is caused by the interaction of several different mechanisms that will be deeply explained later.
Some researchers have simulated the extreme mechanical stimulation provided by the MV on isolated and cultured ASM cells in order to avoid mistakes correlated to the deformational strains induced by the surrounding tissues and to investigate the muscular contractility. They have found raises in cell myosin light chain kinase amount accompanied by increased phosphorylation of the myosin light chain, all key steps in the ASM contractile response32.
The effects of MV are also considered sufficient to cause the most part of the symptoms related to another serious and complex pathology, the broncopulmonary dysplasia (BPD). The patophysiology of this disease will be discussed in the next paragraphs but it is important to underline that the pathogenic processes presided by MV side effects are difficult to distinguish from those which are linked to the oxidative stress caused by hyperoxic exposure.
Both, MV and hyperoxia provoke the epithelium’s inflammation and influence the AW cells' metabolism determining AW remodeling.
Besides, the MV has a direct influence on the cellular inflammation process, inducing the synthesis of cytokines and the radicals’ release from several cell types. These phenomena aggravate the pulmonary inflammation, cause events of apoptosis and affect the cells’ repair mechanisms which have an important role in maintaining the lung architecture and function33.
In conclusion, we can affirm that although MV represent an essential help for the preterm patients, it is necessary to perform further studies in order to refine its management in the clinical practice and elaborate new ventilation strategies able to minimize the unwanted and serious outcomes described above.
22
1.3 Respiratory Disorders in Preterm Infants
Bronchopulmonary dysplasia (BPD) is one of the worst consequences of a PTB. It is a chronic disease that affects the lungs and cause relevant problems to their future development. This disorder is even further problematic because it continues to develop after birth by causing repetitive injuries to the respiratory system.
Bronchial hyperresponsiveness (BHR) and asthma have always been linked together. Despite a great quantity of studies reporting in vivo and ex vivo data, there are still concerns on how these pathologies develop and affect our body.
While there is an extended knowledge about lung damages and the possible ways to face them, little is known about the physiopathological paths.
The understanding of their behavior and effects is a key point for our research as we are studying and evaluating what this type of disorders causes to the respiratory system of preterm infants.
In this chapter there is a general description of BPD, BHR and asthma, their characteristics and physiopathology.
After that it is discussed what are the sequele of prematurity and BPD in newborns and adults. Both rpematurity and BPD are among the main factors that cause the development of asthma, BHR and other severe impairments to the respiratory system.
23
1.3.1 Bronchopulmonary dysplasia (BPD) Definition and history
Bronchopulmonary dysplasia (BPD) is a type of disease which develops as a consequence of injuries beginning with MV of the preterm infants.
It was first described in 1967 by Northway et. al who studied the AW injury and parenchymal fibrosis in preterm babies who were being ventilated. They described the disease as chronic pulmonary disorder occurring in preterm infants with severe respiratory distress syndrome and who have been exposed to aggressive MV and high oxygen concentration.
In those years and approximately before 90s, this disorder affected modestly preterm babies and led to death, then with the use of positive end expiratory pressure (PEEP) ventilation, antenatal steroids and surfactant, survival increased. Yet, many babies who were able to survive, then developed BPD. The use of surfactant, in particular, gave a great spark as it allowed a sensible increase of survival for extremely low weight infants.
Indeed, even if the rate of survival of extremely preterm infants has dramatically increased over years, the prevalence of BPD is reportedly not diminished34.
Often BPD is divided into an old and new version. Referring to the period when no surfactant or steroid treatment were present, BPD is usually addressed as old BPD, which occurred on large preterm infants and was due to heavy ventilation causing barotrauma and pulmonary hypertension. It led to focal septal fibrosis scarring, squamous metaplasia, bronchiolar distortion, mucosal inflammation and fibrosis.
Instead, the term new BPD addresses extremely low birth weight infants and is milder than the old type thank to all the progresses in the medical techniques. It leads to mild epithelia lesions and peribronchial inflammation and fibrosis, fewer and larger simplified alveoli that cause reduced gas-exchange surface (see Fig 1.14).
24 Fig. 1.14: Differences between old and new BPD.
25
Diagnosis, etiology, physiopathology
Nowadays the definition of this disorder is still misleading and not complete, as it is based on the level of support required by infants at 36 weeks post-menstrual age. The disease is present if infants require supplemental oxygen at age 36 weeks post-menstrual age and have required it for the first 28 days of life. A supplemental definition including the severity of the disorder is based on the quantity of oxygen required at 36 weeks GA.
The 10% of very low and the 40% of extremely low birth weight infants develop BPD and there are approximately 5,000 to 10,000 new cases each year in United States only35.
As etiology BPD displays pulmonary immaturity, oxygen toxicity, barotrauma, volutrauma, vitamin deficiency, failed antioxidant protection, excess fluid administration, patent ductus arteriosus (PDA) and chronic infection.
To understand properly the pathophysiology of BPD it has to be taken into account that at about 24 weeks gestation the lung is still quite behind in the growth as it has saccular terminal structures that then segment about six times before alveolarisation, which starts around 32 weeks GA and it is not completed until 36 weeks of age. All these changes and growth have to take place even though preterm delivery happens. Since BPD occurs mostly in preterm infants born at or below 29 weeks GA, the normal alveolar and distal vascular development are interrupted and injury is often superimposed.
These features are well showed in Fig.1.15 which is taken from a study made by Coalson JJ et al. On the left there is a lung tissue section from an infant who was born at 28 weeks GA and treated with prenatal steroids and postnatal surfactant. The tissue has been recovered from an open lung biopsy performed 8 months after birth. The image on the right is from a lung tissue section from a term plus 5 months child. The differences are evident as in the first image enlarged airspaces are clear; some are alveolar ducts (AD) that show scattered alveolar structures (arrow) lacking additional branching. In the second one there are numerous crests and alveolar structures within the airspaces and ducts.
