Stroke lesion outcome prediction based on MRI imaging combined with clinical information

In developed countries, the second leading cause of death is stroke, which has the ischemic stroke as the most common type. The preferred diagnosis procedure involves the acquisition of multi-modal Magnetic Resonance Imaging. Besides detecting and locating the stroke lesion, Magnetic Resonance Imaging captures blood flow dynamics that guides the physician in evaluating the risks and benefits of the reperfusion procedure. However, the decision process is an intricate task due to the variability of lesion size, shape, and location, as well as the complexity of the underlying cerebral hemodynamic process. Therefore, an automatic method that predicts the stroke lesion outcome, at a 3-month follow-up, would provide an important support to the physicians' decision process. In this work, we propose an automatic deep learning-based method for stroke lesion outcome prediction. Our main contribution resides in the combination of multi-modal Magnetic Resonance Imaging maps with non-imaging clinical meta-data: the thrombolysis in cerebral infarction scale, which categorizes the success of recanalization, achieved through mechanical thrombectomy. In our proposal, this clinical information is considered at two levels. First, at a population level by embedding the clinical information in a custom loss function used during training of our deep learning architecture. Second, at a patient-level through an extra input channel of the neural network used at testing time for a given patient case. By merging imaging with non-imaging clinical information, we aim to obtain a model aware of the principal and collateral blood flow dynamics for cases where there is no perfusion beyond the point of occlusion and for cases where the perfusion is complete after the occlusion point.AP was supported by a scholarship from the Fundacao para a Ciencia e Tecnologia (FCT), Portugal (scholarship number PD/BD/113968/2015). This work is supported by FCT with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 Programa Operacional Competitividade e Internacionalizacao (POCI) with the reference project POCI-01-0145-FEDER-006941. We acknowledge support from the Swiss National Science Foundation - DACH320030L_163363

Multicentre evaluation of MRI variability in the quantification of infarct size in experimental focal cerebral ischaemia

Ischaemic stroke is a leading cause of death and disability in the developed world. Despite that considerable advances in experimental research enabled understanding of the pathophysiology of the disease and identified hundreds of potential neuroprotective drugs for treatment, no such drug has shown efficacy in humans. The failure in the translation from bench to bedside has been partially attributed to the poor quality and rigour of animal studies. Recently, it has been suggested that multicentre animal studies imitating the design of randomised clinical trials could improve the translation of experimental research. Magnetic resonance imaging (MRI) could be pivotal in such studies due to its non-invasive nature and its high sensitivity to ischaemic lesions, but its accuracy and concordance across centres has not yet been evaluated. This thesis focussed on the use of MRI for the assessment of late infarct size, the primary outcome used in stroke models. Initially, a systematic review revealed that a plethora of imaging protocols and data analysis methods are used for this purpose. Using meta-analysis techniques, it was determined that T2-weighted imaging (T2WI) was best correlated with gold standard histology for the measurement of infarctbased treatment effects. Then, geometric accuracy in six different preclinical MRI scanners was assessed using structural phantoms and automated data analysis tools developed in-house. It was found that geometric accuracy varies between scanners, particularly when centre-specific T2WI protocols are used instead of a standardised protocol, though longitudinal stability over six months is high. Finally, a simulation study suggested that the measured geometric errors and the different protocols are sufficient to render infarct volumes and related group comparisons across centres incomparable. The variability increases when both factors are taken into account and when infarct volume is expressed as a relative estimate. Data in this study were analysed using a custom-made semi-automated tool that was faster and more reliable in repeated analyses than manual analysis. Findings of this thesis support the implementation of standardised methods for the assessment and optimisation of geometric accuracy in MRI scanners, as well as image acquisition and analysis of in vivo data for the measurement of infarct size in multicentre animal studies. Tools and techniques developed as part of the thesis show great promise in the analysis of phantom and in vivo data and could be a step towards this endeavour

Characterization of microvascular stress and cell death responses triggered by renal ischemia-reperfusion injury and their roles in progressive fibrosis

L’insuffisance rénale aiguë (IRA) est une complication clinique associée à une mortalité significative. Parmi les diverses causes d'IRA, l'ischémie-reperfusion (IRI) est une étiologie importante, en particulier dans le contexte de la transplantation rénale. Les types de mort cellulaire programmée (MCP) activées dans l'IRA induite par IRI ont été étudiées par des nombreux groupes. L’atteinte tubulaire épithéliale est classiquement considérée comme le principal contributeur à l'IRA.En effet, plusieurs morts programmées de cellules tubulaires ont été démontrées dans la littérature. Cependant, les lésions endothéliales microvasculaires rénales attirent davantage l'attention en tant qu'inducteurs cruciaux de dysfonctionnement microvasculaire et de fibrose rénale progressive. Ainsi, certaines équipes de recherche, dont la nôtre a rapporté le développement de l'apoptose endothéliale rénale en association avec l’IRI. Le but de mon travail était donc de caractériser les types de mort cellulaire microvasculaires secondaires à l’IRI et leur contribution à la dysfonction rénale. Pour évaluer l'importance de l'apoptose dans l'IRA induite par IRI, nous avons utilisé un modèle murin d’IRI chez des souris caspase-3 knock-out (KO) et sauvages, avec clampage de l'artère rénale pendant 30 minutes (modèle IRA légère) ou 60 minutes (modèle IRA sévère). Dans le modèle IRA légère, notre résultat montre que la carence en caspase-3 empêche la mort apoptotique des cellules endothéliales dans toutes les phases de l'IRA, atténuant la raréfaction microvasculaire, le dépôt de collagène et la fibrose rénale. L’absence de caspase-3 favorise aussi le maintien d’une perméabilité endothéliale microvasculaire normale à long terme. Toutefois, l’invalidation de la caspase-3 aggrave la mort cellulaire tubulaire à court terme en favorisant la nécroptose, mais améliore l’homéostasie tubulaire à long terme grâce à la préservation des capillaires péritubulaires (PTCs) permettant un maintien de la perfusion tubulaire. En outre, le déficit en caspase-3 est également associé à un effet protecteur contre la raréfaction microvasculaire rénale, la fibrose rénale progressive, ainsi qu'une perméabilité endothéliale améliorée et une préservation de la fonction rénale dans le modèle d’IRA sévère. En conclusion, nos résultats démontrent l'effet crucial de l’apoptose endothéliale microvasculaire en tant qu'inducteur de dysfonctionnement microvasculaire rénal, de raréfaction microvasculaire et de fibrose rénale progressive dans la physiopathologie de l'IRA légère et sévère induite par l'IRI. Ils établissent aussi l’importance prédominante de l’atteinte microvasculaire plutôt que tubulaire épithéliale dans la prédiction de la perte de fonction rénale à long terme suite à une IRI.Acute kidney injury (AKI) is a crucial clinical event, with increasing incidence and mortality. Among various pathogenesis of AKI, ischemia-reperfusion injury (IRI) is an important etiology, especially in the renal post-transplant scenario. The complex of programmed cell deaths (PCD) developed in IRI-induced AKI has been proven in a number of investigations. Renal tubular epithelial injury has been considered as the major contributor in AKI and multiple programmed tubular epithelial cell (TECs) deaths have been demonstrated in the literature. However, renal microvascular endothelial injury is attracting more attention as an important inducer of microvascular dysfunction and renal progressive fibrosis. Some investigators, including our team, have reported the development of renal endothelial apoptosis in the condition of ischemia. Apoptosis, a commonly known programmed cell death, has been elucidated in both renal TECs and microvascular endothelial cells (ECs) post-IRI and the activation of caspase-3 functions as the key effector of caspase-dependent apoptosis. To verify the importance of apoptosis in IRI- induced AKI, we applied the in vivo murine renal IRI model in wild-type and caspase-3 KO mice, with clamping the renal artery for 30 minutes (mild AKI model) or 60 minutes (severe AKI model). In regard to the mild AKI model, our result demonstrates that caspase-3 deficiency prevents ECs apoptotic death in all phases of AKI, attenuating microvascular rarefaction, collagen deposition, and renal fibrosis, while maintaining physical endothelial permeability in the long-term. Meanwhile, caspase-3 deletion aggravates tubular injury in the short-term by promoting TECs necroptosis but ameliorates long-term tubular injury through preserved peritubular capillaries (PTCs) function. Furthermore, caspase-3 deficiency also demonstrated a protective effect against renal microvascular rarefaction, progressive renal fibrosis, as well as enhanced endothelial permeability in the severe AKI model. Conclusively, our findings determine the crucial effect of microvascular endothelial apoptosis as an inducer of renal microvascular dysfunction, microvascular rarefaction, and progressive renal fibrosis in the pathophysiology of mild and severe AKI induced by IRI. Additionally, our results demonstrate the predominant importance of microvascular endothelial injury over tubular epithelial injury in predicting renal function loss at long-term post-IRI

Regulation of pericyte contractility in health and disease

Pericytes regulate blood flow by constricting and dilating capillaries, especially in the brain, which requires a controlled supply of oxygen and energy substrates, and where the majority of the vascular resistance is in the capillaries. This thesis explores cerebral pericyte contractility in health and disease. I showed that pericytes can be identified reliably by bright-field imaging and that the pericyte-specific dye Neurotrace 500/525 in fact preferentially labels pericytes on higher capillary branch orders. Raising pericyte [Ca2+]i evokes a maximum capillary constriction near pericyte somata where pericyte circumferential processes originate, but adjustment of capillary wall tone by longitudinal processes of pericytes on higher order capillary branches may also regulate blood flow. In Alzheimer’s disease (AD) pericytes contract and reduce brain blood flow. The involvement of reactive oxygen species in amyloid β (Aβ)-induced pericyte contraction was assessed. I demonstrated that NADPH oxidase 4 (NOX4) and hydroxyl radicals mediate this process. A combination of NOX4 and endothelin A receptor blockers, or C-type natriuretic peptide, prevented Aβ-induced constriction, suggesting therapeutic approaches to AD targeted at pericytes. Hyperoxia often occurs in clinical situations. In rodent and human brain slices, I showed that hyperoxic perfusion causes capillaries to constrict and pericyte [Ca2+]i to increase. In rat but not human pericytes, this process was inhibited by blocking 20-HETE production, suggesting a role of 20-HETE in hyperoxic pericyte contraction. Cerebral blood flow decreases in COVID-19 patients, in which the SARS-CoV-2 virus binds to angiotensin converting enzyme 2 (ACE2). In the brain I showed that ACE2 is mainly expressed in pericytes. I found that the receptor binding domain (RBD) of the SARS-CoV-2 spike protein potentiates the capillary constriction evoked by angiotensin II, by reducing ACE2 activity, leading to more activation of AT1 receptors by angiotensin II. My results suggest that pericytes could play a role in the neurological complications of COVID-19 and that the AT1R blocker losartan might prevent this

Overcoming conventional modeling limitations using image- driven lattice-boltzmann method simulations for biophysical applications

The challenges involved in modeling biological systems are significant and push the boundaries of conventional modeling. This is because biological systems are distinctly complex, and their emergent properties are results of the interplay of numerous components/processes. Unfortunately, conventional modeling approaches are often limited by their inability to capture all these complexities. By using in vivo data derived from biomedical imaging, image-based modeling is able to overcome this limitation. In this work, a combination of imaging data with the Lattice-Boltzmann Method for computational fluid dynamics (CFD) is applied to tissue engineering and thrombogenesis. Using this approach, some of the unanswered questions in both application areas are resolved. In the first application, numerical differences between two types of boundary conditions: “wall boundary condition” (WBC) and “periodic boundary condition” (PBC), which are commonly utilized for approximating shear stresses in tissue engineering scaffold simulations is investigated. Surface stresses in 3D scaffold reconstructions, obtained from high resolution microcomputed tomography images are calculated for both boundary condition types and compared with the actual whole scaffold values via image-based CFD simulations. It is found that, both boundary conditions follow the same spatial surface stress patterns as the whole scaffold simulations. However, they under-predict the absolute stress values approximately by a factor of two. Moreover, it is found that the error grows with higher scaffold porosity. Additionally, it is found that the PBC always resulted in a lower error than the WBC. In a second tissue engineering study, the dependence of culture time on the distribution and magnitude of fluid shear in tissue scaffolds cultured under flow perfusion is investigated. In the study, constructs are destructively evaluated with assays for cellularity and calcium deposition, imaged using µCT and reconstructed for CFD simulations. It is found that both the shear stress distributions within scaffolds consistently increase with culture time and correlate with increasing levels of mineralized tissues within the scaffold constructs as seen in calcium deposition data and µCT reconstructions. In the thrombogenesis application, detailed analysis of time lapse microscopy images showing yielding of thrombi in live mouse microvasculature is performed. Using these images, image-based CFD modeling is performed to calculate the fluid-induced shear stresses imposed on the thrombi’s surfaces by the surrounding blood flow. From the results, estimates of the yield stress (A critical parameter for quantifying the extent to which thrombi material can resist deformation and breakage) are obtained for different blood vessels. Further, it is shown that the yielding observed in thrombi occurs mostly in the outer shell region while the inner core remains intact. This suggests that the core material is different from the shell. To that end, we propose an alternative mechanism of thrombogenesis which could help explain this difference. Overall, the findings from this work reveal that image-based modeling is a versatile approach which can be applied to different biomedical application areas while overcoming the difficulties associated with conventional modeling

Novel clinical and etiopathogenetic findings in Pseudoxanthoma elasticum

Soft tissue calcification in the human body can be considered part of a process of continuous  degeneration  which  we  tend  to  designate  as  “aging”.  Being  an  example  of technological wit and superb bio-engineering second to none, even the decay of this corpus can hardly be considered a random or passive event. On the contrary, calcium precipitation is regulated quite tightly by an intruiging interplay between stimulatory proteins and inhibitory factors. Thus, it has been foreseen man not to be turned into a chalk pillar in his prime years, but rather to endure a much slower process of gradual mineralization. But when this brilliant regulatory opus starts failing, the reign of human pathology is entered, confronting the body with ectopic mineralization disorders. One of the archetypes of such disease is pseudoxanthoma elasticum or PXE, in which ectopic mineralization of elastic fibres causes skin, ocular and cardiovascular complications. Despite its identification more than two centuries ago, PXE has – as many genetic disorders – always been surrounded by a haze of mystery. It is the aim of this thesis to contribute to the clinical, molecular and histopathological characterization of this fascinating disease. Through careful characterization of the PXE patient cohort followed at the Ghent Center for Medical Genetics, we were able to emphasize important clinical features, such as stroke and peripheral artery disease, as well as identifying novel phenotypical features in patients and carriers, among which were abdominal calcifications and testicular microlithiasis. Also the question of a limited or subclinical phenotype in PXE carriers was addressed and we showed them to be more prone to cardiovascular disease, next to limited ophthalmological symptoms represented by comets and comet tails. In an exploratory pilot study among over 200 consecutive ischemic stroke patients, ABCC6 hotspot analysis yielded a significant increase in ABCC6 mutations compared to a healthy reference population. This signified another example of heterozygous carriers being prone to cardiovascular and/or cerebrovascular disease and introduced the ABCC6 gene in stroke research. In single and multi-center studies, this thesis contributed to the characterization and expansion of the ABCC6 mutation spectrum, as well as the exclusion of genotype-phenotype correlations. The applied molecular strategy for mutation analysis of the ABCC6 gene proved to be an efficient and cost-effective method, yielding the highest mutation detection rate so far. Also, the continuous discussion on the mode of inheritance and in particular the existence of an autosomal dominant form of PXE could be addressed constructively. Throughout the clinical follow-up of PXE patients, we applied novel fundus imaging techniques, such as autofluorescence and infrared imaging, with substantial improvement of the diagnostic capacities of limited or subtle lesions in fundo. Through collaborative efforts, the importance of electrophysiological abnormalities – subdivided in three retinopathy phenotypes – was brought to attention. Within the span of this PhD thesis, a novel phenotype was identified and characterized both clinically and molecularly. This novel autosomal recessive disorder was coined the PXE-like syndrome, because of its resemblance with classic PXE, and was proven to be caused by mutations in the GGCX gene. Encoding the gamma-carboxylase, an enzyme important in the vitamin K (VK)-cycle, this observation implicated VK and proteins depending on this vitamin – among which are several inhibitors of mineralization – in the pathogenesis of the PXE-like syndrome and hence PXE. Through various immunohistochemical and ELISA methods, VK-dependent inhibitors of calcification were shown to be inactive or defective in these syndromes, leading to ectopic mineralization in the PXE-like syndrome but also in PXE patients. These observations could be attributed to the GGCX mutations in the PXE-like syndrome. The observation of extremely low VK serum levels – an essential co-factor for protein carboxylation in the VK-cycle – in PXE patients explained why the VK-cycle is defective in PXE. The exact link with the impaired ABCC6 transporter remains unclear, although it is tempting to think of VK or one of its associated molecules as the substrate of ABCC6. Also, these findings hold out the prospect of VK suppletion as a treatment for PXE. As such, the findings summarized in this thesis have elaborated the clinical and molecular knowledge of PXE and related disorders, and have opened novel avenues for further fundamental and applied research in the field of ectopic mineralization. Above all, they have benefitted patients and their family though a more efficient molecular diagnosis, a more to-the- point follow-up and the prospect of a treatment for their burdensome disease

Mitochondrial Dysfunction in Ageing and Diseases

The past decade has witnessed an explosion of knowledge regarding how mitochondrial dysfunction may translate into ageing and disease phenotypes, as well as how it is modulated by genetic and lifestyle factors. Impairment of the mitochondria may be caused by mutations or deletions in nuclear or mitochondrial DNA. Hallmarks of mitochondrial dysfunction include decreased ATP production, decreased mitochondrial membrane potential, swollen mitochondria, damaged cristae, increased oxidative stress, and decreased mitochondrial DNA copy number. In addition to energy production, mitochondria play an important role in regulating apoptosis, buffering calcium release, retrograde signaling to the nuclear genome, producing reactive oxygen species (ROS), participating in steroid synthesis, signaling to the immune system, as well as controlling the cell cycle and cell growth. Dysfunctional mitochondria have been implicated in ageing and in several diseases, many of which are age-related, including mitochondrial diseases, cancers, metabolic diseases and diabetes, inflammatory conditions, neuropathy, and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease. Additionally, a possible link between mitochondrial metabolism and the ubiquitin-proteasome and autophagy-lysosome systems is emerging as a novel factor contributing to the progression of several human diseases. This special issue calls for original research, mini and full reviews, and perspectives that address the progress and current standing in the vast field of mitochondrial biology. These include, but are not limited to: ageing neurodegenerative diseases mitochondrial diseases metabolic diseases protein homeostasis cell/retrograde signaling oxidative stress pain cancer immune system therapies to counteract mitochondrial dysfunctio