Cirrhosis is a detrimental condition of the liver, o.en attributed to an unhealthy lifestyle (e.g. alcohol abuse and fatty diet). It is characterized by severe distortion of the hepatic architecture, impairing the hepatic perfusion and function. Once cirrhosis reaches the decompensated stage, i.e. when complications become clinically present, it may culminate into liver insu.ciency or cancer. Cirrhosis is ranked the twel.h leading cause of death worldwide, with mortality rates exceeding a million deaths in óþÕþ. Cirrhosis is still considered irreversible and early aetiological treatment focuses on delaying disease progression and reducing complications. However, liver transplantation remains the only therapeutic option when liver insu.ciency and/or complications occur. Hence, it has become imperative to precisely assess the patient’ liver (function) to tailor the treatment. However, a patient-centred therapeutic approach remains challenging as the pathogenesis of cirrhosis is still not fully understood. As disruption and remodelling of the hepatic vasculature is recognized a key factor of cirrhogenesis, this work aimed at clarifying the intricate relation between the progression of the disease, the hepatic (angio)architectural disarrangement, and the impaired vascular perfusion. is dissertation is divided into three major parts. Part I gives a background overview of cirrhosis, while part II focuses on the original research conducted within the framework of this PhD. Part III concludes this dissertation by describing the key ťndings and a number of interesting prospects for future research. This part provides a background overview on the healthy liver, the cirrhotic liver, and the state-of-the-art techniques in cirrhosis modelling.

Chapter Õ.e healthy liver

The liver is the largest internal organ in humans, performing over ¢þþ metabolic and detoxifying functions. Its vascular system is uniquewith regard to other organs because of its dual blood supply: the hepatic artery provides the liver with oxygenated blood, whereas the portal vein delivers nutrientenriched but partially oxygen-depleted blood. Both vessels run in parallel throughout the liver’ interior and divide repeatedly through several orders of branches until reaching the terminal ramiťcations, the so-called portal triads. Blood guided through these portal triads is mixed in the sinusoids, where the molecular exchange with the parenchymal liver cells occurs.e blood stream continues down the sinusoids until collected in the central veins, which join to form hepatic veins and ensure liver drainage. e hepatic microarchitecture consists of ťve cellular components. Hepatocytes are the parenchymal cells, making up .þÛ of the liver tissue. e remaining cell types, i.e. sinusoidal endothelial cells, Kup.er cells, hepatic stellate cells, and pit cells, are harboured in the sinusoids. e cellular cross-talk between the group of sinusoidal cells and hepatocytes is critical to maintain body homeostasis and physiological processes such as liver regeneration. As these complex communication pathways are tightly regulated, abnormalities in intercellular communication are increasingly being recognized to underlie virtually every liver disease.

Chapter ó.e cirrhotic liver

Cirrhosis is deťned as the common end-point of any chronic liver disease. Its pathogenesis results from repetitive destruction and regeneration of hepatocytes. is relentless process disturbs the hepatic architecture, eventually impairing the hepatic perfusion and liver function. Cirrhosis is o.en attributed to alcohol abuse, viral infections, or metabolic syndromes related to obesity and diabetes.ese causes may elicit the development of chronic liver diseases, such as fatty liver disease or chronic viral hepatitis, and the consecutive progression to cirrhosis. With progression to cirrhosis, the hepatic architecture is gradually replaced by di.use ťbrosis, vascularized ťbrous septa, and regenerative nodules.ese morphological features mainly result from overly exuberant wound-healing mechanisms, activated to repair the hepatocellular damage. One of these mechanisms is ťbrogenesis, i.e. the production of ťbrous tissue to limit and encapsulate the injured area. However, the ongoing damage leads to the exorbitant deposition of this high-density ťbrous tissue. As a consequence, septa may develop to form ťbrous bridges between portal triads and central veins. Blood vessels embedded in these septa become isolated from the parenchyma and may transform into widened shunt vessels. ese low-resistance blood channels bypass and impoverish the hepatocytes of nutritive blood, contributing to the hepatocellular necrosis. e latter instigates the uncontrolled proliferation of the remaining hepatocytes, leading to the formation of regenerative nodules. e excessive ťbrous tissue and regenerative nodules severely compress the hepatic blood vessels and, as such, increase the overall intrahepatic vascular resistance to blood .ow. Dynamic vascular changes (i.e. sinusoidal remodelling and angiogenesis) also contribute to the progressive increase of intrahepatic vascular resistance, eventually leading to portal hypertension. Portal hypertension is the earliest and most common complication of cirrhosis. It is characterized by an elevated portal pressure due to the obstruction of portal .ow. Many of the complications (e.g. ascites and variceal haemorrhage) related to cirrhosis commence in the setting of worsening portal hypertension. Treatment of cirrhosis is therefore directed at lowering the portal pressure, delaying the disease progression, or suppressing complications. For advanced stages of cirrhosis, liver transplantation remains the only available therapeutic option. Early and accurate detection has thus become imperative for e.ective treatment or reversal of the progressive liver disease. However, the latter remains a complex issue, as the pathogenesis of cirrhosis is still not fully understood.

Chapter ì. Techniques to model cirrhosis

Over the past decades, a plethora of modelling techniques has been applied to gain more insight into the pathogenesis of cirrhosis. Animal models (e.g. mice, rats, and pigs) allowed researchers to revisit the process of cirrhogenesis in a controlled way. Note, however, that a model re.ecting all characteristics/stages of human cirrhosis is yet to be developed. Currently, three rodent models are widely used to induce cirrhosis: the carbon-tetrachloride model, the chronic bile duct ligation model, and the thioacetamide model. In this dissertation, we chose the thioacetamide rat model to mimic cirrhogenesis because of its documented reliability and reproducibility. Besides animalmodels, several experimentalmethods have been adopted tomeasure the hepatic haemodynamics or functional/mechanical parameters of the (cirrhotic) liver in vivo. Invasive methods (e.g. the microspheres technique and the multiple-indicator dilution technique) were mostly applied to quantify the altered hepatic (micro)haemodynamics in animals, while non-invasive methods (e.g. intravital .uorescence microscopy and elastography techniques) also allowed for assessment of morphological derangements and alteredmechanical properties of liver tissue (e.g. sti.ness). Interestingly, experimental data not only increased our understanding of the pathogenesis of cirrhosis, but also provided fundamental information to develop quantitative computational models. Available computational models have focused on the adaptive hepatic vasculature, impaired liver perfusion, or liver dysfunction in the case of cirrhosis. Two experimental approaches (i.e. vascular corrosion casting and immunohistochemistry) were frequently used to study the altered branching pattern and/or geometrical features of the hepatic vasculature for varying animal species. e hepatic perfusion was generally assessed based on computational .uid dynamics (CFD) models and electrical analog models. Models of liver (dys)function, on the other hand, applied a multiscale modelling approach to integrate biological processes into perfusion systems. ey allowed for prediction of the dynamic hepatic response a.er liver damage (e.g. liver regeneration), or the risk of (long-term) liver injury a.er environmental exposure to chemical substances. While the aforementioned research solved some pieces of the puzzle, more information is needed to reveal the complete picture and understand the complex pathogenesis of cirrhosis. This part focused on elucidating the intricate relation between the progression of the disease, the hepatic (angio)architectural disarrangement, and the impaired vascular perfusion using an established rat model of cirrhosis.

Chapter ¦. ìD reconstruction of the rat hepatic vasculature

Since relatively little was known about the topographical organisation of the rat hepatic vasculature, a methodological framework was developed and implemented to quantitatively analyse and model the vasculature of rat livers across multiple scales. In this chapter, the framework was developed and optimised for healthy rat livers. e framework comprised two experimental techniques, i.e. vascular corrosion casting (VCC) and immunohistochemistry (IHC), to acquire morphological data on the hepatic vasculature. While both techniques were previously used to study the hepatic vasculature, they still faced a number of challenges. We optimised these techniques and combined their complementary strengths to reconstruct in ìD the rat hepatic circulation across multiple scales. e VCC and micro-CT scanning protocol was improved by enabling dual casting via the hepatic artery and portal vein, whereas the IHC was extended with an adapted clearing technique (CUBIC) to allow for deep tissue imaging when combined with confocal microscopy. Using so.ware developed in-house, the vascular network - in both VCC and IHC datasets - was automatically segmented and/or morphologically analysed. Hence, the framework allowed for detailed ìD visualization of the hepatic vasculature and automated quantiťcation of the morphological parameters and branching topology.

Chapter ¢. Vascular remodelling of the rat liver during cirrhogenesis

Knowledge on the dynamically evolving pathological changes of the hepatic vasculature during cirrhogenesis is limited. More speciťcally, detailed morphological data of the vascular adaptations during disease development is lacking. In this chapter, we addressed this lacuna through quantiťcation of the rat hepatic macro- and microvasculature at di.erent time points during cirrhogenesis. Cirrhosis was chemically induced using the thioacetamide (TAA) rat model. At four time points (þ, ä, Õó, and Õ. weeks), the hepatic vasculature was ťxed and visualized using a combination of vascular corrosion casting and deep tissue microscopy (optimised in chapter ¦). ìD reconstruction and data ťtting enabled cirrhogenic features to be extracted at multiple scales, portraying the impact of cirrhosis on the hepatic vasculature. At the macrolevel, we noticed that regenerative nodules severely compressed pliant venous vessels fromÕóweeks of TAAintoxication onwards. Especially, hepatic veinswere highly a.ected by this compression, with collapsed vessel segments severely reducing perfusion capabilities. At the microlevel, we discovered zone-speciťc sinusoidal degeneration with sinusoids located near the surface being more a.ected than those in the middle of a liver lobe. e datasets shed more light on the evolving angioarchitecture during cirrhogenesis and formed the basis of the computational model as presented in chapter ä.

Chapter ä. Modelling rat haemodynamics during cirrhogenesis

Cirrhosis is characterized by severe distortion of the hepatic architecture and mechanical properties (chapter ¢). e architectural disarrangements progressively increase the intrahepatic vascular resistance, leading to portal hypertension and systemic circulatory disorders. In this chapter, we assessed the impact of these changing vascular resistances on the hepatic and global circulation haemodynamics during cirrhogenesis. Morphological quantiťcation of the branching topology of the hepatic vascular trees, as generated in chapter ¢, provided the input for a lobe-speciťc lumped parameter model of the liver. is liver model was coupled to a closed-loop model of the entire circulation of the rat.e integrated model allowed hepatic and systemic haemodynamics to be predicted for di.erent stages of cirrhogenesis. e simulations showed the e.ect of the altering hepatic vascular resistances (driven by the hepatic venous resistance increase) on the haemodynamics, with portal hypertension observed a.er Õó weeks of TAA intoxication. Moreover, the lobe-speciťc liver model revealed abnormal .ow patterns (e.g. reversal of portal venous .ow) in the diseased animals.ese phenomena are not uncommon in patients with cirrhosis. e closed-loop model was further extended to account for compensation mechanisms and disorders of the systemic circulation, frequently observed in cirrhosis. eir impact on the hepatic, systemic, and pulmonary haemodynamics was simulated. Results clearly explained how cirrhosis-induced vascular changes severely disrupt both hepatic and global haemodynamics. Since the model is able to simulate the main characteristics of cirrhosis, it may be translated to humans for the assessment of liver interventions (e.g. transjugular intrahepatic portosystemic shunt (TIPS) surgery).

Chapter ß. Conclusions and future perspectives

A key achievement of this dissertation is the development and implementation of a methodological framework to quantify the vascular remodelling of the rat liver during cirrhogenesis. is allowed novel and unique ìD morphological data to be generated on the hepatic macro- and microcirculation throughout cirrhosis development.ese data then formed the basis for a computationalmodel to simulate the rat blood circulation during cirrhogenesis. is computational model allowed for assessment of the haemodynamic consequence of cirrhogenesis and provided unique insights into the manifestation of portal hypertension. We consider our work a step forward in the unravelling of the complex pathogenesis of cirrhosis. However, a lot of ground is yet to be uncovered. An interesting ťeld for future research may be the integration of functional models into the presented computational model to study the interplay between the hepatic perfusion and liver function in the case of cirrhosis.