Quantification of Pulmonary Ventilation using Hyperpolarized 3He Magnetic Resonance Imaging

Smoking-related lung diseases including chronic obstructive pulmonary disease (COPD) and lung cancer are projected to have claimed the lives of more than 30,000 Canadians in 2010. The poor prognosis and lack of new treatment options for lung diseases associated with smoking are largely due to the inadequacy of current techniques for evaluating lung function. Hyperpolarized 3He magnetic resonance imaging (MRI) is a relatively new technique, and quantitative measurements derived from these images, specifically the ventilation defect volume (VDV) and ventilation defect percent (VDP) have the potential to provide new sensitive measures of lung function. Here, we evaluate the reproducibility of VDV, and explore the sensitivity of these measurements in healthy young and elderly volunteers, and subjects with smoking-related lung disease (COPD and radiation-induced lung injury (RILI)). Our results show that 3He MRI measurements of ventilation have high short-term reproducibility in both healthy volunteers and subjects with COPD. Additionally, we report that these measurements are sensitive to age-related changes in lung function. Finally, in RILI we show that measurements of lung function derived from 3He MRI are sensitive to longitudinal changes in lung function following treatment, while in COPD we report that using VDP in conjunction with structural measurements of disease (using the apparent diffusion coefficient (ADC) derived from diffusion-weighted images) may provide a new method for phenotyping this smoking-related lung disease

Pulmonary Image Segmentation and Registration Algorithms: Towards Regional Evaluation of Obstructive Lung Disease

Pulmonary imaging, including pulmonary magnetic resonance imaging (MRI) and computed tomography (CT), provides a way to sensitively and regionally measure spatially heterogeneous lung structural-functional abnormalities. These unique imaging biomarkers offer the potential for better understanding pulmonary disease mechanisms, monitoring disease progression and response to therapy, and developing novel treatments for improved patient care. To generate these regional lung structure-function measurements and enable broad clinical applications of quantitative pulmonary MRI and CT biomarkers, as a first step, accurate, reproducible and rapid lung segmentation and registration methods are required. In this regard, we first developed a 1H MRI lung segmentation algorithm that employs complementary hyperpolarized 3He MRI functional information for improved lung segmentation. The 1H-3He MRI joint segmentation algorithm was formulated as a coupled continuous min-cut model and solved through convex relaxation, for which a dual coupled continuous max-flow model was proposed and a max-flow-based efficient numerical solver was developed. Experimental results on a clinical dataset of 25 chronic obstructive pulmonary disease (COPD) patients ranging in disease severity demonstrated that the algorithm provided rapid lung segmentation with high accuracy, reproducibility and diminished user interaction. We then developed a general 1H MRI left-right lung segmentation approach by exploring the left-to-right lung volume proportion prior. The challenging volume proportion-constrained multi-region segmentation problem was approximated through convex relaxation and equivalently represented by a max-flow model with bounded flow conservation conditions. This gave rise to a multiplier-based high performance numerical implementation based on convex optimization theories. In 20 patients with mild- to-moderate and severe asthma, the approach demonstrated high agreement with manual segmentation, excellent reproducibility and computational efficiency. Finally, we developed a CT-3He MRI deformable registration approach that coupled the complementary CT-1H MRI registration. The joint registration problem was solved by exploring optical-flow techniques, primal-dual analyses and convex optimization theories. In a diverse group of patients with asthma and COPD, the registration approach demonstrated lower target registration error than single registration and provided fast regional lung structure-function measurements that were strongly correlated with a reference method. Collectively, these lung segmentation and registration algorithms demonstrated accuracy, reproducibility and workflow efficiency that all may be clinically-acceptable. All of this is consistent with the need for broad and large-scale clinical applications of pulmonary MRI and CT

¹⁹F-MRI of inhaled perfluoropropane for quantitative imaging of pulmonary ventilation

PhD ThesisMRI of exogenous imaging agents offers a safely repeatable modality to assess regional pulmonary ventilation. A small number of studies have validated the safety and potential utility of 19F imaging of inhaled thermally polarised perfluoropropane. However, the relative scarcity of signal in restrictive breath hold length acquisition times inhibits translation of this technique to clinical application. This work presents methods used to maximise the attainable image quality of inhaled perfluoropropane. Novel quantitative measures of ventilation and perfusion have been investigated and discussed. A preliminary healthy volunteer study was conducted to verify the efficacy of the imaging technique and to assess perfluoropropane wash-in and wash-out rates. Quantitative assessment of the suitability of four RF coil designs was performed, comparing power efficiency with loading and signal homogeneity within the sensitive volume of each coil. The 3D spoiled gradient echo sequence was simulated, accounting for the power performance of the chosen birdcage coil design, for calculation of acquisition parameter values required to achieve the highest SNR in a fixed acquisition period for 19F-MRI of inhaled perfluoropropane. Studies on resolution phantoms and healthy volunteers assessed the performance of the optimised imaging protocol, in combination with a compressed sensing technique that permitted up to three-fold acceleration. Two novel lung-representative phantoms were fabricated and used to investigate the behaviour of the MR properties of inhaled perfluoropropane with changing structural and magnetic environments. Finally, a method for lengthening the T2* of inhaled perfluoropropane by susceptibility matching the alveolar tissue to the inhaled gas by intravenous injection of a highly paramagnetic contrast agent is presented. Initial development work was conducted in phantoms and rodents before translation to healthy volunteers. This technique offers the potential to concurrently acquire images reflecting both pulmonary ventilation and perfusion

Development and evaluation of new inspiratory muscle loading technologies for use in inspiratory muscle training

Inspiratory muscle training (IMT) is becoming more popular in sporting populations as contentious issues associated with the magnitude of its effect as an ergogenic training aid are gradually dispelled. The main reason for this is the increasing good practice in controlled intervention studies particularly through the use of appropriate outcome measures, for example time trial performance, but also the work done to identify potential mechanisms such as the blood flow redistribution model. The purpose of this thesis is to further improve the benefits afforded by IMT through the development and evaluation of new technologies. The key aim was to produce technologies that provide functional relevance to a sporting population by permitting normal, albeit loaded ventilation to take place in an ambulatory situation. A number of intermediate objectives have been achieved including the identification of the key limitations of existing technologies and their application, the development of new methods for the prescription of a dynamic inspiratory load via a series of human studies i.e. mouth pressure generation due to inspiratory drive during exercise, 3-Dimensional thoracic displacement and peripheral thoracic force generation, and the design, manufacture and evaluation of two new inspiratory muscle training technologies. These are a thoracic restricting technology that provides true ambulatory loading and a variable mouth occlusion technology that can be set to load in accordance with the specific pressurevolume characteristics of an individual. The former (thoracic restricting technology) has been developed into a first stage prototype and tested on a single subject to assess any changes to breathing pattern. The results suggest that suitable load location may minimise any adverse effects and has enabled further theoretical development to take place. The latter (variable mouth occlusion technology) has been implemented in a controlled study on a group of healthy male adults to assess its functionality and the suitability of a specific decaying load. The results suggest that the chosen load may have been unsuccessful in increasing the work of breathing and that specific aspects of the functionality require development thus enabling the selection of specific refinements for future interventions to be identified. The focus of future research is therefore the practical comparison of these new technologies with existing devices in order to fully understand the optimisation of inspiratory muscle training

Validation of volatile organic compounds for the assessment of liver disease

Chronic liver disease is one of the few conditions with increasing morbidity and mortality rates. Up to 75% of individuals with cirrhosis are diagnosed during a decompensation episode, at which point the prognosis is poor. Cirrhotic patients also have an annual risk of 2 to 4% of developing hepatocellular carcinoma (HCC). HCC is currently the fourth leading cause of cancer-related mortality worldwide, which is at least in part due to late diagnosis and inadequate screening. Gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOCs) in breath has the potential to form the basis of a non-invasive diagnostic test for chronic liver disease and HCC. However, exhaled VOCs can be influenced by multiple confounding factors and the equipment used to collect and analyse breath can be cost prohibitive. The aims of my PhD were four-fold. Firstly, to develop and validate a novel, cost-effective breath collection device and to formulate a standard operating procedure for its use in clinical studies. Secondly, to analyse the VOC profile of background room air within common clinical sampling locations and to assess their potential impact upon the collection of breath samples. Thirdly, to investigate a methodology for sample splitting using GC-MS as a way of facilitating sample analysis across multiple mass spectrometry platforms. With the information garnered from this methodology work, my final aim was to perform a clinical study to profile the VOCs in the exhaled breath of patients with cirrhosis, HCC, and normal liver parenchyma. Prior to this, I also performed a critical analysis of the pre-existing literature on VOCs for assessment of liver disease to help guide my study design. Analysis of the novel breath collection device revealed acceptable repeatability for a wide range of VOCs and optimum settings for flow rates and volumes of breath were determined and included within a standard operating procedure. Profiling the background air volatiles in sampling locations identified specific VOC signatures for each location. Breath samples did not separate by location but monitoring of background volatiles in parallel to breath sampling remains important for identification of contaminant VOCs. Splitting of desorbed breath samples via GC-MS and recollection of two samples back on to one thermal desorption tube provides the best discrimination between samples. For my main clinical study, breath samples of 149 patients were analysed using GC-MS. Elevated levels of limonene and 2-pentanone were identified in those with hepatopathology, validating the results of previous studies. Additional VOCs were also discovered as candidate biomarkers and further studies are required to validate these findings. The results of my clinical study have added to the existing literature that specific VOCs in exhaled breath have the potential to form a non-invasive diagnostic test for hepatopathology that could potentially help enhance earlier diagnosis of liver disease and reverse the trend in mortality rates.Open Acces

Evaluating technologies in the assessment of pulmonary disease to aid lung transplantation: from ex vivo to in vivo models

INTRODUCTION: Despite technological advances, the rate of utility of potential lung donors for transplantation remains low. As a consequence, more than a third of patients listed for lung transplant will not receive one. Application of emerging technologies to select suitable donor organs may help to increase the donor organ pool. Those who receive a lung transplant remain at risk of significant morbidity and mortality and there is a lack of useful biomarkers to identify those at risk of rejection. In this thesis I aim to explore the use of new technologies currently not part of standard practice in the UK to improve the potential donor pool for transplant by (i) evaluating an ex vivo lung perfusion (EVLP) platform to physiologically assess lungs declined for transplant; (ii) developing a novel method for assessing pulmonary vascular leak using EVLP and optical fibre- based endomicroscopy; and (iii) using the optical fibre-based endomicroscopy platform in a first in human study to assess in situ enzyme activity which may be implicated in graft rejection. METHODS: Retrospective study of a national database of multiorgan donors. Prospective assessment of human lungs declined for transplant using a custom built EVLP platform. Fibered endomicroscopy in ex vivo and in vivo animal models of acute lung injury. First in human study of fibered endomicroscopic molecular imaging of in situ matrix metalloprotease (MMP) activity. RESULTS: Lung transplantation from multiorgan donors remains low in Scotland, usually due to suboptimal functional assessment. Our results suggest that further isolated ex vivo assessment may offer additional confidence in graft function. I demonstrate that it is possible to image leaked intravascular protein in situ using an ex vivo perfused acute lung injury model, however we were unable to translate this into an in vivo porcine acute lung injury model. I show that it is possible to optically image, in real-time, pulmonary enzyme activity using a bespoke molecular smartprobe and to observe drug-target engagement with an enzyme inhibitor. DISCUSSION: For those with life limiting pulmonary disease, lung transplantation may offer the only opportunity for survival. Despite increasing operative rates world-wide the vast majority of donated lungs are deemed unsuitable and as a result more than one third of those listed never receive a transplant. Those that do remain at risk of subsequent morbidity and mortality. In order to improve the rate of transplant as a lifesaving intervention and reduce the post-operative risk to recipients a rapid adoption of technological innovation is required to better characterise the lung both pre and post transplant

Pulmonary lipid homeostasis in cigarette smoke-associated lung diseases

Introduction. Les effets du tabagisme demeurent problématiques dans notre société. Les mécanismes initiateurs de la réponse immunitaire pulmonaire induits par la fumée de cigarette sont peu caractérisés. Un des phénomènes dominants en contexte tabagique est l’augmentation de la taille des macrophages alvéolaires. Ce changement phénotypique se distingue par une accumulation intracellulaire de lipides suggérant que le transport lipidique pulmonaire des macrophages alvéolaires est modifié en contexte tabagique. Le transport lipidique pulmonaire est composé de plusieurs étapes, dont la capture, le remaniement et l’export de lipides par les macrophages alvéolaires. Les impacts du tabagisme sur le transport lipidique pulmonaire sont actuellement inconnus. Hypothèse. Le tabagisme altère le transport lipidique pulmonaire. Objectifs : Chapitre 1) Investiguer l’impact de l’exposition à la fumée de cigarette sur le transport lipidique pulmonaire dans un modèle murin et chez l’humain ainsi qu’évaluer l’impact d’une thérapie d’augmentation des high-density lipoproteins (HDLs) dans un modèle murin. Chapitre 2) Investiguer les effets d’une thérapie avec un agoniste du récepteur nucléaire liver X receptor (LXR) activant l’export lipidique dans un modèle murin. Chapitre 3) Caractériser les répercussions d’une carence alimentaire sur la santé pulmonaire et sur la réponse pulmonaire en contexte tabagique dans un modèle murin. Méthodes. 1. Le transcriptome pulmonaire de souris exposées à la fumée de cigarette et de sujets non-fumeurs, fumeurs et ex-fumeurs a été étudié. La capacité d’efflux de cholestérol a été mesurée dans le sérum et dans le lavage bronchoalvéolaire (LBA) de souris fumeuses et non fumeuses. Une HDL recombinante, le MDCO-216, a été administrée à des souris fumeuses et non-fumeuses et leurs réponses immunitaires, leurs fonctions pulmonaires et leurs compositions corporelles ont été analysées. 2. Un agoniste du LXR, le T0901317, a été administré à des souris en contexte tabagique. Le transcriptome pulmonaire relié au transport lipidique, la réponse immunitaire pulmonaire et du macrophage alvéolaire ainsi que les impacts sur le surfactant pulmonaire ont été investigués. 3. Des souris fumeuses et non fumeuses ont été nourries avec des diètes déficientes en méthionine (MD), choline (CD) et méthionine et choline (MCD) et leurs fonctions pulmonaires, leurs réponses immunitaires pulmonaires et leurs expressions géniques pulmonaires ont été caractérisées. Résultats. Chapitre 1. L’expression des gènes impliqués dans le transport lipidique pulmonaire murin et humain est modifiée en contexte tabagique. La capacité du sérum et du LBA à promouvoir la sortie de cholestérol est augmentée après une seule exposition à la fumée de cigarette. L’administration du MDCO-216 réduit la réponse inflammatoire pulmonaire et la taille des macrophages alvéolaires dans un modèle d’exposition aiguë à la fumée de cigarette. Le MDCO-216 semble protéger les fonctions pulmonaires et induit une augmentation de la quantité de masse maigre chez les souris fumeuses. Chapitre 2. L’agoniste du LXR augmente l’expression des gènes de transport lipidique pulmonaire, cependant il exacerbe la réponse immunitaire pulmonaire en contexte tabagique. Les macrophages alvéolaires ont aussi un phénotype inflammatoire exacerbé et ont davantage de stress au réticulum endoplasmique lorsqu’ils sont traités en contexte tabagique. L’activation de LXR mène à une réduction des niveaux de surfactant pulmonaire. Chapitre 3. La diète MCD altère les fonctions pulmonaires en induisant un profil restrictif pulmonaire et abolit la réponse immunitaire pulmonaire à la fumée de cigarette. En histologie, ces souris nourries avec la diète MCD n’ont toutefois aucun foyer fibrotique pulmonaire. L’expression génique de plusieurs gènes associés à la matrice extracellulaire et les niveaux de surfactant pulmonaire sont réduits chez les souris nourries avec la diète MCD. Les phénotypes pulmonaires de la diète MCD sont toutefois réversibles après un retour d’une semaine sur la diète contrôle. La diète CD induit un profil pulmonaire de type emphysémateux et la diète MD mène à un profil restrictif. Conclusions. Ces travaux démontrent que le transport lipidique pulmonaire a un rôle majeur en contexte tabagique et qu’il est modulé rapidement. La thérapie d’augmentation des HDLs, avec le MDCO-216, propose une nouvelle voie de traitement envisageable pour les ex-fumeurs. La thérapie ciblant LXR suggère qu’il pourrait y avoir des effets délétères chez les sujets fumeurs actifs. Les carences alimentaires en méthionine et en choline démontrent d’importants changements sur la physiologie pulmonaire. Ce tout nouveau domaine de recherche, le nutri-respiratoire, requiert davantage d’études afin de mieux comprendre l’impact d’une mauvaise nutrition sur la santé pulmonaire.Introduction. Cigarette smoking remains a major problem in our society.While a lot of cigarette smoke impacts are actually known, few data are available on initiating mechanisms involved in the pulmonary immune response to cigarette smoke. One of the most intriguing phenomena under cigarette smoke exposure conditions is the presence of enlarged alveolar macrophages. This phenotypic change is characterized by an intracellular lipid accumulation which may be a sign of inadequate lipid export by alveolar macrophages induced by cigarette smoking. Pulmonary lipid transport begins with lipid capture, lipid reorganization and lipid droplet formation followed by lipid export by alveolar macrophages. Cigarette smoke impacts on these steps are actually unknown. Hypothesis. Cigarette smoking alters pulmonary lipid transport. Objectives: Chapter 1) To investigate the effect of cigarette smoke exposure on pulmonary lipid transport in cigarette smoke-exposed mice and in healthy controls, smokers and former smokers. To investigate the impact of high-density lipoprotein (HDLs) therapeutic potential in cigarette smoke-exposed mice. Chapter 2) To investigate, in mice, the therapeutic potential of an agonist activating the nuclear receptor liver X receptor (LXR) involved in the transcription of lipid export genes. Chapter 3) To explore, in mice, if a dietary deficiency alters the pulmonary health and the pulmonary response to cigarette smoke. Methods. 1. The pulmonary transcriptome of cigarette smoke-exposed mice and healthy controls, smokers and former smokers was assessed. Cholesterol efflux capacity of serum and bronchoalveolar lavage (BAL) was measured in unexposed and cigarette smoke-exposed mice. MDCO-216, a recombinant HDL, was administered to unexposed and cigarette smoke-exposed mice and analyzed their pulmonary immune response, lung functions and body composition. 2. T0901317, an LXR agonist, was systemically given to mice under cigarette smoke exposure conditions. Pulmonary genes associated with lipid transport, lungs and alveolar macrophage immune pulmonary response to cigarette smoke and the impact of T0901317 on the pulmonary surfactant were assessed. 3. Unexposed and cigarette smoke-exposed mice were fed with methionine deficient (MD), choline deficient (CD) or methionine and choline deficient (MCD) diet. Diets impact on lung functions, pulmonary immune response to cigarette smoke and pulmonary transcriptome were characterized. Results. Chapter 1. Cigarette smoking altered the expression of pulmonary lipid transport genes in mice and in humans. Serum and BALF cholesterol efflux capacities were increased following a twohour cigarette smoke exposure. MDCO-216 dampened the pulmonary inflammatory response and reduced the size of alveolar macrophages in our acute cigarette smoke exposure model. MDCO-216 also seemed to be beneficial to lung functions and induced an increase in lean mass in cigarette smokeexposed treated mice. Chapter 2. T0901317 treatments led to an increase in the expression of pulmonary lipid transport genes. However, it also induced an exacerbated pulmonary immune response during cigarette smoking. Cigarette smoke-exposed treated-alveolar macrophages displayed an exacerbated inflammatory phenotype and showed an augmented endoplasmic reticulum stress. Furthermore, LXR activation led to pulmonary surfactant depletion under cigarette smoke exposure conditions. Chapter 3. The MCD diet altered lung function displaying a restrictive profile and almost abolished the pulmonary immune response to cigarette smoke. Lung histology showed no signs of fibrosis, a phenotype usually associated with restrictive pulmonary functions. MCD diet led to a dramatic change in the pulmonary expression of extracellular matrix genes and also reduced pulmonary surfactant levels. Nevertheless, these pulmonary phenotypes were reversible within a week when mice were refed a control diet. Interestingly, the CD diet induced an emphysema-like profile, while MD diet showed similar pulmonary functions to the MCD diet. Conclusions. The present thesis adds major data to an underestimated field of research and demonstrates the importance of pulmonary lipid transport, especially during cigarette smoking. Recombinant HDL therapy with MDCO-216 may be a new opportunity to overcome adverse effects of cigarette smoking, while activating LXR seems rather deleterious. Nutrient deficiencies, such as methionine and choline led to unprecedented impacts on the pulmonary health and on the pulmonary response to cigarette smoke. This completely new field of research, “nutri-respiratory”, requires additional studies to fully decipher the impact of unhealthy nutrition on the respiratory system

Quantitative lung CT analysis for the study and diagnosis of Chronic Obstructive Pulmonary Disease

The importance of medical imaging in the research of Chronic Obstructive Pulmonary Dis- ease (COPD) has risen over the last decades. COPD affects the pulmonary system through two competing mechanisms; emphysema and small airways disease. The relative contribu- tion of each component varies widely across patients whilst they can also evolve regionally in the lung. Patients can also be susceptible to exacerbations, which can dramatically ac- celerate lung function decline. Diagnosis of COPD is based on lung function tests, which measure airflow limitation. There is a growing consensus that this is inadequate in view of the complexities of COPD. Computed Tomography (CT) facilitates direct quantification of the pathological changes that lead to airflow limitation and can add to our understanding of the disease progression of COPD. There is a need to better capture lung pathophysiology whilst understanding regional aspects of disease progression. This has motivated the work presented in this thesis. Two novel methods are proposed to quantify the severity of COPD from CT by analysing the global distribution of features sampled locally in the lung. They can be exploited in the classification of lung CT images or to uncover potential trajectories of disease progression. A novel lobe segmentation algorithm is presented that is based on a probabilistic segmen- tation of the fissures whilst also constructing a groupwise fissure prior. In combination with the local sampling methods, a pipeline of analysis was developed that permits a re- gional analysis of lung disease. This was applied to study exacerbation susceptible COPD. Lastly, the applicability of performing disease progression modelling to study COPD has been shown. Two main subgroups of COPD were found, which are consistent with current clinical knowledge of COPD subtypes. This research may facilitate precise phenotypic characterisation of COPD from CT, which will increase our understanding of its natural history and associated heterogeneities. This will be instrumental in the precision medicine of COPD

T cell targeted nanoparticles for pulmonary siRNA delivery as novel asthma therapy

The aim of this work was to develop and optimize a T cell targeted delivery system for pulmonary delivery of siRNA directed against GATA3, the central transcription factor of Th2 cytokines, as a novel therapy for asthma. Therefore, an existing carrier system on the basis of polycationic polymer polyethylenimine (PEI) and targeting Ligand transferrin (Tf), resulting in the so-called Tf-PEI, was chosen and fully characterized concerning relevant siRNA polyplex characteristics such as size, zeta potential, siRNA encapsulation efficiency and gene silencing capability in vitro and in vivo. Subsequently, Tf-PEI was blended with a second conjugate, Tf-Mel, containing the lysosomal Peptide melittin, in order to increase endosomal escape of the polyplexes. Resulting Tf-Mel-PEI blends were characterized and optimized to achieve siRNA polyplexes combining specific targeting of activated T cells and efficient cytoplasmic siRNA release, resulting in successful gene knockdown. For GATA3 silencing, a suitable siRNA sequence combination was found and applied within the Tf-Mel-PEI blend polyplexes to investigate down-stream effects of the gene knockdown on cytokine levels. These were concludingly tested in an optimized model for activated T cells as a first step for evaluation of relevant therapeutic effects in an inflammatory environment