The renin angiotensin system (RAS) exerts many diverse physiological functions throughout the body, mediated by its effector peptide, angiotensin II (AngII). AngII has been linked with a variety of different functions ranging from the initiation of severe vascular pathologies, such as atherosclerosis and abdominal aortic aneurysm (AAA), to mundane physiological processes of fluid homeostasis, vascular contraction, and regulation of blood pressure. To provide a potential link between these functions, an in-depth analysis of regional effects of AngII on aortic vasculature was performed. The studies presented in this dissertation tested the overall hypothesis of whether regional changes exist in the vasculature in response to angiotensin II (AngII). We first infused AngII into C57BL/6 animals and studied the aortic morphology in detail. On first glance, we detected a thickening throughout the aorta, with no overt changes from region to region. However, upon further analysis, it was demonstrated that there was a region-specific aortic arch hyperplasia, versus the hypertrophy in the remainder of the aorta. Through a series of experiments, this hyperplasia was linked to the redox-mediated protein Id3. Further analysis of the vasculature demonstrated AngII exerted aortic contractions which were limited to the infrarenal aorta. These contractions were mediated by the AT1b receptor subtype in the RAS. We also demonstrate that AngII leads to suprarenal specific formation of AAA, which can be attenuated by the deletion of specific innate immune mediator proteins, such as MyD88 and TLR4. Overall, these data suggest many region-specific roles for AngII in the aortic vasculature and provide many novel findings as to the cause of these effects.

The overall aim of this thesis is to explore experiences and self-reported outcomes from Transcatheter Aortic Valve Implantation, TAVI, among people with severe aortic stenosis. The thesis includes four studies. Study I-II are based on interviews performed the day before TAVI and Qualitative Concept Analysis was used for analysis. Study III is based on interviews at six months’ follow-up and Grounded Theory was used for analysis. Study IV is quantitative and based on questionnaires at baseline and at six months’ follow-up. Nonparametric, descriptive statistics were used for the analysis. Study I described the vulnerable situation for patients with severe aortic stenosis before TAVI. They were facing death and at the same time struggling to cope with their symptoms and to maintain independent. TAVI offered hope but also caused uncertainty about the new method. Study II focused on the patients’ decision-making process. Three patterns were identified; ambivalent, obedient, and reconciled. The ambivalent patient is unsure of the value of treatment and aware of the risks; the obedient patient is unsure of the value of one's own decision and wants to leave the decision to others; the reconciled patient has reached a point where there is no choice anymore and is always sure that the decision to undergo TAVI is right. Study III offered a deeper understanding of the TAVI trajectory. A journey of balancing between hope and life-struggle was the core category of the analysis. Before TAVI patients felt threatened, but also experienced hope. The rehabilitation phase was described as demanding and depressing or surprisingly simple. At the six months’ followup patients described being pleased to return to life, however, many were still struggling with limitations. Study IV focused on quantifying the symptom burden, function and health related quality of life before and after TAVI. The results were reflected against that of patients treated with open surgery. Self-rated function and health related quality of life increased and symptoms were reduced at follow-up, but breathlessness and fatigue were still common. Conclusively, TAVI patients are struggling with limitations, both because of their comorbidities and because of their valve disease which also poses a threat to their lives. TAVI gives an opportunity to survive, to stay independent and to increase quality of life. To feel and preserve hope is essential for patients’ wellbeing, both before and during the recovery process.

Background: Aortic valve insufficiency (AI) at the time of left ventricular assist device (LVAD) insertion needs to be corrected, however there is little known about how to manage bioprosthetic valvular AI. Case presentation: A 55-year-old female with dilated cardiomyopathy who previously had a bioprosthetic aortic valve replacement needed a LVAD as a bridge to transplant. Her left ventricular ejection fraction was 10% and had mild to moderate transvalvular AI. She underwent a HeartWare HVAD insertion along with aortic valvular coaptation stitch repair (Park's stitch) to the bioprosthetic valve. Conclusion: Her AI improved to trivial with minimal ejection through the bioprosthetic valve. She was transplanted 6 months following the surgery. A Park's stitch to the bioprosthetic aortic valve with more than mild AI might be a good option for bridge to transplant patient.

Calcific aortic valvular disease (CAVD) is the most common valvular pathology in the developed world. CAVD results in calcifications forming on the aortic valve leaflets, inhibiting proper closure and causing complications of stenosis and regurgitation. Although, the mechanisms behind the disease initiation are unknown, it is believed to be a cell-mediated phenomenon, and not the result of passive degradation of the valve as once believed due to the increased prevalence with age. Currently, there are no pharmaceutical options for the prevention or reversal of calcifications, the only treatment option is complete valve replacement, an imperfect solution. Hindering the development of potential therapeutics is that currently there are no adequate animal models which replicate the calcification and cell death seen in disease explanted valves. An in vitro model has been develop where valvular interstitial cells (VICs), the main cell type of the valve, are seeded at high density into tissue culture polystyrene dishes and cultured with TGF-β1. This results in VICs activating to the myofibroblast phenotype and forming cell aggregates. Due to currently unknown mechanisms, apoptosis occurs within the center of the aggregates and calcification ensues. Although simplistic, this model has been used to show that rate and frequency of aggregation is affected by cellular tension; conditions of high tension increase aggregation response, while conditions of low tension prevent aggregation and calcification from occurring. It is important to note; however, that despite its wide usage, the current model is limited as the aggregation and subsequent calcification are random occurrences and are not consistent across literature where same conditions for control samples are used. The motivation of the presented work is two-fold. First, high intracellular tension has been suggested as one of the mechanisms leading to disease in the valve. Despite the clear and important role of cell tension, VIC tension has never before been measured in a dynamic environment. The ways in which dynamic stimulation affects individual VIC tension is not known. In aim one, a method is developed to allow for long-term cyclic stretch of VICs with measurement of cell traction force. It was found that cyclic stretch decreased cell tension in cells with high prestress and increased cell tension for conditions of low prestress. Combined, these findings indicate a homeostatic cellular tension which is dependent upon the mechanical environment. In the second aim, a novel method for creating VIC aggregates is validated. Micro-contact printing, essentially “stampingâ€� of a protein in a defined pattern, is used to create circular aggregates on polyacrylamide gels. This method allows for the separation of the aggregation from the subsequent calcification, an improvement over the current in vitro model. The method is then used to explore the role of the distribution of tension in the initiation of disease

Endovascular aortic repair (EVAR) is a minimally invasive alternative to open surgery for the treatment of aortic aneurysms (AA). However, standard EVAR is not applicable to complex AA with involvement of vital branches, which could be occluded by the endograft. As an emerging technique, the concept of multiple overlapping uncovered stents (MOUS) have been proposed to manage complex lesions. MOUS was used to modulate the flow pattern inside the aneurysm sac, and promote the thrombus formation followed by the aneurysm shrinkage. In this dissertation, we sought to investigate the mechanism of MOUS-induced flow modulation and key factors associated with the success of this novel technique: - The mechanical behaviour of AA was characterised by uniaxial material tests (Chapter 4). A Bayesian framework was proposed for material constants identification. They were found correlated to the microstructure of tissue fibre network and were capable in differentiating tissue types. - Solid-to-solid interaction and one-way fluid-solid interaction (FSI) analysis was performed based on patient-specific computer tomography angiography (Chapters 5&6). Structural stress concentrations were observed within the landing zones, which increased with the number of stents deployed. In the parameter studies (Chapter 6), the overall porosity was identified as the dominant factor of the flow-diverting outcome, while cross-stent structures of MOUS had limited influence. - The pathological effect of structural stress concentration induced by an implanted device was further studied in rabbit models (Chapter 7). The wall structural stress and fluid shear stress were obtained from FSI analysis based on magnetic resonance imaging (MRI), and correlated to plaque characteristics. Both high structural stress and low fluid shear stress were found correlated to plaque initialisation and increased inflammation. Overall, MOUS modulates the blood flow with robust performance under different overlapping patterns. Image-based biomechanical analysis can optimise MOUS design and can contribute to personalised pre-surgery planning.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the developed world, claiming almost 17,000 deaths annually in the United States. The lack of noninvasive therapeutics to slow or halt the disease warrants the need for further understanding of the pathobiological mechanisms of CAVD. A tri-laminar structure of aortic valve determines the biomechanical properties of its leaflets. Valvular endothelial cells (VECs) and interstitial cells (VICs) are responsible for valve structural integrity. Traditional two-dimensional culture conditions spontaneously activate the pathological differentiation of VICs making in vitro studies challenging. A monolayered three-dimensional (3D) hydrogel platform was recently developed as a novel in vitro culture system to study the phenotypic changes of VICs leading to microcalcification (early stages of calcification). This system, however, did not fully recapitulate the microenvironment of native valve tissues because of the lack of individual layer representations and endothelial coverage. Bioprinting technology, which allows precise and integrated positioning of cells, matrix, and biomolecules, may provide an innovative approach toward building a more biologically relevant 3D culture platform. OBJECTIVE: This study aims to lay the groundwork for building a multilayered 3D-bioprinted culture platform to study CAVD by first validating the use of bioprinting in monolayered cell-laden 3D hydrogel constructs. METHODS: Human VICs were isolated from patients undergoing valve replacement surgeries at Brigham and Women’s Hospital (Boston, MA) according to Institutional Review Board (IRB) protocols. VICs were expanded in culture medium containing growth factors for up to 6 passages and then encapsulated in hydrogels using 3D bioprinting technology. After encapsulation, VIC-laden 3D constructs were cultured in either normal or osteogenic conditions for 21 days. Microcalcification, cell proliferation, and cell apoptosis were evaluated using fluorescent staining and confocal microscopy. Results were compared with results from VIC-laden hydrogels made manually. RESULTS: An increase in microcalcification was observed throughout bioprinted VIC-laden hydrogel constructs cultured in osteogenic conditions for 21 days, whereas normal conditions developed negligible calcification signals. Cell proliferation and apoptosis were not significantly different between normal and osteogenic groups in bioprinted hydrogels. Cell-free hydrogels did not exhibit any microcalcification. Overall, bioprinted hydrogels showed less nonspecific background staining than handmade hydrogels, thus providing a better means for quantitative assessments of 3D culture platforms. CONCLUSION: Based on bioprinting technology, an improved monolayered cell-laden hydrogel platform was successfully established as a first step toward building an in vitro multilayered disease model for studying the pathobiological mechanisms of CAVD. The results in this study were consistent with current literature that proposes calcification as a cell-dependent, apoptotic-independent, and proliferation-independent pathway. / 2019-07-13T00:00:00Z

Le rétrécissement aortique calcifié dégénératif (RAC) représente la principale pathologie valvulaire cardiaque dans les pays occidentaux (2% à 7% de la population après 65 ans). Il n’existe, à ce jour, aucun traitement médical qui permette de stopper ou de prévenir la progression du RAC. Le seul traitement du RAC sévère est le remplacement valvulaire aortique chirurgical ou par cathétérisme (TAVI) en cas de symptômes (dyspnée,douleur thoracique ou syncope), de dysfonction ventriculaire gauche (FEVG<50%) ou de mauvaise tolérance fonctionnelle lors d’un test d’effort. À l’inverse, la prise en charge des patients asymptomatiques avec un RAC sévère est controversée devant le risque de mort subite ou de détérioration myocardique irréversible sans traitement et le risque de la chirurgie cardiaque prophylactique et des complications prothétiques. De plus, le RAC intéresse une population de plus en plus âgée où la présence de symptômes peut être difficilement évaluable et où s’associent des facteurs de risques et des comorbidités cardiovasculaires rendant la stratégie clinique de plus en plus compliquée. Enfin, il semble que parmi les patients avec un RAC serré asymptomatique, certains sont à plus haut risque d’évènements et bénéficieraient peut être d’une chirurgie plus précoce. L’identification de marqueurs de progression et pronostiques objectifs dans ce sous-groupe de patients constitue donc un enjeu très important. / Aortic valve stenosis (AS) is the most common valvular heart disease in Westerncountries AS (2%–7% of the population after 65 years old) for which valve replacement is theonly curative treatment. Current guidelines recommend surgery in patients with severe ASand either symptoms or left ventricular systolic dysfunction (LVEF<50%). However,treatment of asymptomatic patients remains controversial due, on the one side, to the riskof sudden death without preceding symptoms and irreversible myocardial dysfunction and,on the other side, to the risk of surgery and prosthetic valve complications. Identifyingsubsets of asymptomatic AS patients with preserved left ventricular ejection fraction whomay benefit from early or prophylactic surgery is therefore a critical clinical challenge.

A normal aortic valve has three leaflets; however, 1- 2% of children are born with an aortic valve with two leaflets, referred to as congenital bicuspid aortic valves (BAV). Recent in vivo studies have shown that flow development past the bicuspid valves into the ascending aorta is markedly different from that past the normal tri-leaflet aortic valve. This difference may lead to the bicuspid valve having a higher rate of ascending aortic root dissection, a pathology that can potentially result in fatality. Using computational fluid dynamics we aim to evaluate the alterations in flow development in the ascending aorta with BAV compared to healthy tri-leaflet valves (TAV) and relate the alterations in flow-induced stresses with higher incidences of aortic dissection in patients with BAV. Simplified models based on the geometry and dimensions from published literature were developed. The preliminary results show that there is a difference in flow development between the BAV and the tri-leaflet valve. This is visible by the differences in wall shear stress and dynamic pressure distribution in the ascending aorta. The conclusion drawn from this is that there are marked differences in the ascending aortic flow development with BAV compared to that with TAV which may lead to dissection of the aortic arch.

Experiments described in this thesis address the pathogenesis of aortic valve sclerosis/stenosis using a bench to bedside approach. In particular, the thesis begins with development of a technique using ultrasonic backscatter analyses to quantitate the early stages of aortic stenosis. Subsequent chapters utilized this methodology to quantitate aortic valve structural changes in a model and intervention study of aortic stenosis in rabbits. The last chapters are human studies designed to identify factors associated with presence of aortic sclerosis/stenosis; with particular interest in potential association of endothelial dysfunction/inflammation/platelet aggregation with abnormal aortic valve structure quantitated by ultrasonic backscatter. In Chapter 1 (Introduction) the relevant literature is reviewed. Development of ultrasonic backscatter to quantitate aortic sclerosis (Chapter 2) Aortic valve sclerosis (ASc) is detected when there is visual assessment of focal increases in echogenicity of the aortic valve most commonly assessed by echocardiography. However, there is no previously described method to quantitate degree of aortic valve structural abnormality as ASc is not associated with marked hemodynamic obstruction quantifiable by Doppler echocardiography. The current study used ultrasonic backscatter to quantitate aortic valve structural abnormality in patients assessed as having ASc based on valve appearances, compared to young healthy volunteers with normal aortic valves. The results of the study indicate: 1) that the mean levels of aortic valve backscatter in ASc patients are approximately 60% greater than in young healthy volunteers (ie aortic valve backscatter scores ≥ 16dB are not consistent with normal aortic valve structure), 2) ultrasonic backscatter scores in ASc patients are directly correlated with subjective scoring of sclerosis and with a positive trend with transvalvular pressure gradients in patients with mild-moderate aortic stenosis, and most importantly, 3) ultrasonic backscatter is a reproducible technique, with mean differences between estimates based on repeat echocardiograms of 2.3 ± 1.7 (9.1%). These results indicate that ultrasonic backscatter could be used as a quantitative measure of aortic valve structural abnormality in epidemiology and for examination of interventions. In vivo studies Development of an animal model of aortic stenosis with vitamin D2 (Chapter 3) The aim of the study was to develop an appropriate animal model for AS. The study used vitamin D2 alone at 25,000IU/4 days weekly (vit-D2) for 8 weeks to induce AS in rabbits. Results showed that: 1) rabbits in the vit-D2 group had significantly increased in transvalvular velocity and pressure gradients compared to rabbits in the control group (normal chow + drinking water); this was consistent for aortic valve ultrasonic backscatter scores; 2) aortic valve immunohistochemistry/histology showed marked calcification, neutral lipids, macrophage, and leukocyte infiltrations for rabbits in the vit- D2 group (ie consistent with histology of human AS); 3) significant elevation of asymmetric dimethylarginine (ADMA) concentrations in the vit-D2 group occurred compared to controls over the 8 weeks treatment period; the change in ADMA concentrations correlated significantly with the change in transvalvular pressure gradients for rabbits in the vit-D2 group; 4) rabbits in the vit-D2 group had significantly impaired endothelium-dependent acetylcholine-induced aortic relaxation, and this effect was completely abolished by the nitric oxide synthase inhibitor (L-NAME); 5) the addition of 0.5% cholesterol-supplemented diet to the vitamin D2 regimen did not accentuate the development of AS. Thus, treatment with vitamin D2 at 25,000IU/4 days weekly for 8 weeks significantly induced AS with similar aortic valve pathology to that of human AS; therefore, the model is suitable for use in examining potential therapeutic interventions in AS. Effects of ramipril on development of AS in rabbits (Chapter 4) Using this animal model, this study aimed to examine the effects of the angiotensinconverting enzyme inhibitor (ACEi) ramipril on development of AS. Rabbits (n=28) treated for 8 weeks were divided into 2 groups: (a) vitamin D2 alone (n=10) (normal chow + 25,000IU vitamin D2 in drinking water); (b) vitamin D2/Ramipril (n=12) (normal chow+25,000IU vitamin D2/Ramipril (0.5mg/kg) in drinking water). Six further rabbits constituted a normal reference group (no treatment was given). The results for comparisons between vitamin D2/ramipril vs vitamin D2 alone were as follows: 1) ramipril-treated rabbits had significantly less severe hemodynamic obstructions (p<0.05, for both) as assessed by transvalvular velocity, and aortic valve area; with borderline reduction in aortic valve backscatter (p=0.08); 2) ramipril significantly reduced plasma ADMA concentrations; 3) there was improvement in acetylcholine-induced aortic relaxation (p=0.056), with significant improvement in sodium nitroprusside-induced relaxation (p<0.05); 4) there was a strong inverse correlation between acetylcholineinduced aortic relaxation and aortic valve backscatter score (0<0.001), thus providing further evidence of the potential role of nitric oxide in retarding the development of AS in this model. These data provide a strong rationale for the inception of a randomized trial of ACE inhibition as a strategy for limitation of AS progression in humans. Human studies Aortic stenosis is associated with elevated plasma levels of asymmetric dimethylarginine (ADMA) concentrations in humans (Chapter 5). Given the findings that aortic stenosis induced by vitamin D2 in rabbits also caused elevation of plasma ADMA concentrations, a physiological inhibitor of nitric oxide synthase, a mediator and marker of endothelial dysfunction and an indicator of incremental cardiovascular risk. The study sought to determine whether plasma ADMA concentrations are elevated independently of pre-existing coronary risk factors in subjects with at least moderate aortic stenosis (n=42) compared to age-matched patients with normal aortic valves (n=42): as determined both by visual assessment and with aortic valve backscatter scores < 16dB. Results for this study were as follows: 1) plasma ADMA concentrations were not statistically different between the AS and non-AS group (median 0.59 vs 0.54 µmol/L, p=0.13, Mann-Whitney test) on univariate analysis; 2) backward stepwise multiple linear regression showed the presence of AS was a significant predictor of elevated ADMA concentrations (p=0.04, 95% CI =0.001, 0.072). 3) in addition, elevated plasma ADMA concentrations were also associated with history of atrial fibrillation (p=0.009, 95% CI=0.015, 0.100), and negatively associated with creatinine clearance (p=0.01, 95% CI=-0.002, 0.000), and the use of statin therapy (p=0.01, 95% CI=-0.081, -0.011). Therefore, in conclusion, this study found that AS is independently associated with elevation of ADMA concentrations, beyond that implied by “conventional” risk factors for endothelial dysfunction. The clinical status of AS as an incremental marker of cardiovascular risk may reflect ADMA-mediated endothelial dysfunction. Assessment of factors associated with ASc in a random ageing population study (Chapter 6). There have been few clinical studies of factors associated with ASc. Previous population studies have established that ASc is an independent correlate of incremental risk of coronary events. Having established that patients with AS have increased plasma ADMA concentrations (Chapter 5), it was now aimed to determine whether subjects with increased aortic valve backscatter scores (ASc) also have other markers of endothelial dysfunction/NO effects, independent of preexisting coronary risk factors. The study was designed to identify such anomalies, if they existed, on an incremental basis to other putative correlates of ASc, including coronary risk factors, renal dysfunction and vitamin D levels. Random selected subjects (n=253) aged between 51 to 77 years were evaluated. All patients underwent transthoracic echocardiography examination; aortic valve ultrasonic backscatter score (AVBS), was used to quantitate echogenicity of the aortic valve. Conventional coronary risk factors were identified on history. Integrity of NO generation/response was assessed via (i) plasma ADMA concentrations; (ii) inhibition of platelet aggregation by the NO donor sodium nitroprusside (SNP); (iii) aortic augmentation index (AIx), a measure of arterial stiffness/wave reflection. All putative correlations with AVBS were examined by univariate and stepwise multiple linear regression analyses. On the basis of echocardiographic appearances, ASc was present in 63 subjects (25.4%); mean AVBS scores was 14.9±4.6dB (SD) vs 11.2±3.9dB (SD) in the presence vs absence of ASc (p<0.001). Univariate analyses revealed that platelet responsiveness to NO was inversely correlated with AVBS (β=-0.16, p=0.02); but [ADMA] and AIx were not. On multiple linear regression, significant correlates of increased AVBS were: (i) advanced age (β=0.21, p=0.003), (ii) low body mass index (β=-0.23, p=0.001); and (iii) impaired platelet responsiveness to NO (β=-0.16, p=0.02). In Chapter 7, the implications of the overall findings in this thesis are discussed in relation to future perspective. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309350 / Thesis(Ph.D.) -- School of Medicine, 2008

Introduction: Neurological complications and stroke in association with cardiac surgery is a serious problem. The stroke event can occur during surgery (early stroke) or in the postoperative period with a symptom free interval (delayed stroke). Particle embolization due to aortic manipulation during surgery has been suspected as a mechanism for early stroke. The present thesis address mechanisms and survival effects of stroke both clinically (I-III) and experimentally (IV-V). Methods: Study I) Within a cohort of 2641 consecutive cases, a group of cardiac surgery patients with stroke and evaluated by computed tomography (CT) were studied (n=77). CT-findings were analyzed in relation to stroke symptoms. Study II) Data from 9122 patients undergoing coronary surgery were analyzed. Records of patients with any signs of neurological complications were reviewed to extract 149 subjects with stroke at extubation (early, 1.6%) versus 99 patients having a free interval (delayed, 1.1%). Early and delayed stroke were evaluated separately. Independent risk factors for stroke were analyzed by logistic regression and survival by Cox regression (9.3 years median follow-up). Study III) Patients with early (n=223) and delayed stroke (n=116) were identified among 10809 patients undergoing cardiac and aortic surgery, both groups exposed to cardiopulmonary bypass. Stroke patients were subdivided by the hemispheric location of lesions. Subgroups were compared and their associated pre- and peroperative variables and survival were analyzed. Study IV) Aortic cross-clamp manipulation was studied in a human cadaveric perfusion model. The pressurized aorta was repeatedly cross-clamped and washout samples were collected before and after clamp maneuvers. Particles in the washout samples were evaluated by microscopy and by digital image analysis. Study V) Pig aortas were pressurized and cannulated. Washout samples were collected before and after cannulation (n = 40). Particles were deposited onto a 10-μm filter to be evaluated by microscopy and digital image analysis. Results: Study I) In the group of patients exposed to routine cardiac surgery (i.e., clamping and cannulation) and with early stroke, right-hemispheric lesions were more frequent than of the contra-lateral side (P=0.005). Patients with aortic dissections had a strong dominance of bilateral findings, which was different from the unilateral pattern in the routine-surgery group (P&lt;0.001). Study II) Early and delayed stroke did not share any risk factors. Both early and delayed stroke explained mortality in the early postoperative period (P&lt;0.001, P&lt;0.001 respectively) but also at long term follow-up (P=0.008, P&lt;0.001 respectively). For patients surviving their first postoperative year, delayed but not early stroke influenced long-term mortality (P=0.001 and P=0.695, respectively). Study III) Stroke lesions in association to cardiac surgery were near exclusively ischemic. Early stroke had a preponderance for right-hemispheric lesions (P=0.009). In contrast, patients with early stroke that had undergone surgery of the aorta with circulatory arrest showed a pattern with more bilateral lesions compared to ‘cardiac-type’ operations (P&lt;0.001). Patients with bilateral lesions had a dramatically impaired survival compared to those with unilateral lesions (P&lt;0.001). Study IV) In the cadaveric perfusion model, cross-clamping produced a significant output of particles, which was seen for size intervals of 1 mm and smaller (P=0.002 to P=0.022). In all size intervals the particle output correlated with the degree of overall aortic calcification (P =0.002 to P=0.025). Study V) At cannulation of the pig aorta, more particles were noted after cannulation compared to before the maneuver (P&lt;0.001). This increase included small (&lt;0.1 mm, P&lt;0.001) and intermediate-size particles (0.1-0.5 mm, P&lt; 0.001). Particles above 0.5 mm were few and were not associated with cannulation. Conclusions: The influence of stroke on mortality was devastating, for both early and delayed stroke. These two stroke groups had obvious differences in both their risk factors and their hemispheric distribution. It is here emphasized that early and delayed stroke should be considered as two separate entities with suggested mechanistic differences. Ischemic lesions accounted for near all stroke events seen in association to cardiac surgery. For early stroke, these were mostly located within the right hemisphere. Results from the experimental studies underscore microembolic risks associated with aortic manipulation.