D W Slaaf

Technische Universiteit Eindhoven, Eindhoven, North Brabant, Netherlands

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Publications (187)532.31 Total impact

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    ABSTRACT: The endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 - 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 - 80), which was significantly higher than in B6 bifurcations (0 (0 - 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 μm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 μm in young mice to 3.0 µm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 μm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.
    Full-text · Article · Sep 2011 · Thrombosis and Haemostasis
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    ABSTRACT: The endothelial glycocalyx (EG) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell-vessel wall interactions. To establish an atheroprotective role of the EG, adequate imaging and quantification of its properties in intact, viable, atherogenesis-prone arteries is needed. Carotid arteries of C57Bl6/J mice (n=22) were isolated including the bifurcation, mounted in a perfusion chamber, and perfused with fluorescent lectin wheat germ agglutinin-fluorescein isothiocyanate. The EG was visualized through the vessel wall using two-photon laser scanning microscopy. An image quantification protocol was developed to assess EG thickness, which was sensitive to hyaluronidase-induced changes. In the lesion-protected common carotid artery, EG thickness was found to be 2.3 ± 0.1 μm (mean ± SEM), while the surface area devoid of (wheat germ agglutinin-sensitive) EG was 8.9 ± 4.2%. Data from the external carotid artery were similar (2.5 ± 0.1 μm; 9.1 ± 5.0%). In the atherogenesis-prone internal carotid artery the EG-devoid surface area was significantly higher (27.4 ± 5.5%, p<0.05); thickness at the remaining areas was 2.5 ± 0.1 μm. The EG can be adequately imaged and quantified using two-photon laser scanning microscopy in intact, viable mounted carotid arteries. Spatial EG differences could underlie atherogenesis.
    Full-text · Article · Jun 2011 · Journal of Vascular Research
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    ABSTRACT: MR vessel size imaging (MR-VSI) is increasingly applied to noninvasively assess microvascular properties of tumors and to evaluate tumor response to antiangiogenic treatment. MR-VSI provides measures for the microvessel radius and fractional blood volume of tumor tissue. However, data have not yet been evaluated with three-dimensional microscopy techniques. Therefore, three-dimensional two-photon laser scanning microscopy (TPLSM) was performed to assess microvascular radius and fractional vessel volume in tumor and muscle tissue. TPLSM data displayed a mazelike architecture of the tumor microvasculature and mainly parallel oriented muscle microvessels. For both MR-VSI and TPLSM, a larger vessel radius and fractional blood volume were found in the tumor rim than in the core. The microvessel radius was approximately six times larger in tumor and muscle for MR-VSI than for TPLSM. The tumor blood volume was 4-fold lower with MR-VSI than with TPLSM, whereas muscle blood volume was comparable for both techniques. Differences between the tumor rim, core, and muscle tissue showed similar trends for both MR-VSI and TPLSM parameters. These results indicate that MR-VSI does not provide absolute measures of microvascular morphology; however, it does reflect heterogeneity in microvascular morphology. Hence, MR-VSI may be used to assess differences in microvascular morphology.
    No preview · Article · Apr 2010 · Magnetic Resonance in Medicine

  • No preview · Article · Jan 2010
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    ABSTRACT: In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (approximately 1 microm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET).
    Full-text · Article · Jan 2010 · Journal of Biomedical Optics
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    ABSTRACT: Thrombosis and embolization are main causes of morbidity and mortality. Up to now, the relative importance of mediators involved is only partly known. It was the aim of this study to investigate the involvement of ADP and thrombin in subsequent phases of arteriolar hemostasis and thromboembolism in vivo. Rabbit mesenteric arterioles were punctured, which induced bleeding, hemostasis, and subsequent thromboembolism. This reaction as well as the activation state of platelets involved ([Ca(2+)](i)), was monitored in real time by intravital (fluorescence) microscopy. Neither inhibition of thrombin formation or thrombin activity nor blockade of platelet ADP receptors P2Y(1) and P2Y(12) influenced the initial hemostatic reaction: in all experiments initial bleeding was stopped by a primary thrombus within 2-3 s. On the other hand, both thrombin inhibition and P2Y(1) blockade increased rebleeding frequency, which indicates reduced thrombus stability in the long term. Finally, inhibition of either thrombin or ADP (via both receptors) reduced aggregate formation during the embolization phase by at least 90%. While most participating platelets exhibited a transient increase in [Ca(2+)](i) during embolization, an increased percentage of platelets showed no calcium response at all during P2Y(1) blockade, which was accompanied by reduced platelet-platelet interaction strength. Whereas thrombin and ADP are not involved in the initial hemostatic reaction, both substances appear to be essential to prevent rebleedings in the long term. During subsequent embolization, ADP (via both receptors) and small amounts of thrombin are involved in platelet activation.
    Full-text · Article · Jul 2009 · Microcirculation
  • D W Slaaf · M H P van Genderen
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    ABSTRACT: The development of a fully integrated biomedical engineering programme (life sciences included from the start) is described. Details are provided about background, implementation, and didactic concept: design centred learning combined with courses. The curriculum has developed into a bachelor-master's programme with two different master's degrees: Master's Degree in Biomedical Engineering and Master's Degree in Medical Engineering. Recently, the programme has adopted semester programming, has included a major and minor in the bachelor's degree phase, and a true bachelor's degree final project. Details about the programme and data about where graduates find jobs are provided in this paper.
    No preview · Article · Jun 2009 · Proceedings of the Institution of Mechanical Engineers Part H Journal of Engineering in Medicine
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    ABSTRACT: Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.
    No preview · Article · Mar 2009 · Small
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    ABSTRACT: Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.
    No preview · Article · Jan 2009 · Small
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    ABSTRACT: There are basically two types of branching patterns in the terminal part of the arteriolar tree. On the one hand, in a number of tissues, including the developing chick embryo chorioallantoic membrane (CAM), the pattern is dichotomous, whereas in other tissues many arteriolar-arteriolar connections, arcades, are found. The structure of the branching pattern depends on the local physical and chemical environment. The goal of this study was to investigate whether substances with an effect on vascular growth influence the vascular branching pattern. We treated chick embryo CAMs daily from day 7 to day 14 postfertilization with 0.9% NaCl, angiotensin II (ANG-II), ANG-II in combination with different angiotensin receptor subtype antagonists, i.e., losartan and CGP 42112 A, or the prostaglandin synthesis inhibitor acetylsalicylic acid (ASA). Arcade formation was quantified by counting the number of arcades per cm2 treated area, the branch-node ratio and mean surface area of arcade loops. ANG-II caused a 2-fold increase in the number of arcades versus 0.9% NaCl. Addition of ASA or losartan caused a further enhancement of arcade formation expressed in the number and branch-node ratio. CGP 42112A had no significant effect on arcade formation. From these data we hypothesize that ANG-II stimulates the process of capillary upgrading to arterioles by stimulation of arteriolar smooth muscle cell growth. Prostaglandins normally counteract this effect. After blockade of prostaglandin action, the ANG-II-induced arterialization is enhanced, resulting in pronounced arcade formation. The actions of losartan may be related to its inhibitory effects on prostaglandins rather than angiotensin receptor antagonism.
    No preview · Article · Sep 2008 · Journal of Vascular Research

  • No preview · Chapter · Apr 2008
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    ABSTRACT: We used two-photon laser scanning microscopy (TPLSM) to demonstrate for the first time its potential in studying relational details at the cellular level of atherogenesis in intact, viable mouse carotid arteries. Isolated and mounted arteries of ApoE-/-mice, aged 15 or 21 weeks (7 and 13 weeks on western diet), were imaged after labeling with specific fluorescent markers for cell nuclei, inflammatory cells, collagen, and lipids. Data were compared with C57BL6/J mice fed a chow diet. Control vessels had intact endothelium without adhering blood cells or significant intimal collagen labeling. In ApoE-/-mice already at 15 weeks, inflammatory cells adhered to the endothelium and increased labeling of collagen was observed in tunica intima at both lesion-prone and non-lesion-prone sites, indicating endothelium activation. In plaques, internalized inflammatory cell density increased with age and plaque progression in tunicae adventitia and intima, but not media. In the whole plaque, aging or plaque progression did not alter the direct relationship between inflammatory cells and collagen. However, within the fibrous caps specifically, direct contact between inflammatory cells and collagen increased with age. This study demonstrates the potential of TPLSM in determining detailed information regarding the complex relationship between inflammatory cells and collagen during atherogenesis.
    No preview · Article · Jan 2008 · Journal of Biomedical Optics
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    ABSTRACT: Blood vessels are part of a subtle regulatory system with differential properties along the vascular tree. Alterations in the vessel wall may lead to various diseases such as atherosclerosis. Current knowledge of vascular alterations is mostly based on histological studies of isolated samples that have lost viability. Functional properties of various compounds in the atherosclerotic vessel wall are still largely unknown. Better understanding of the functionality of the atherosclerotic arterial wall, and, thus, increased insight in (development of) atherosclerotic lesions requires studying of these properties in vivo or in viable arteries ex vivo. This talk focuses on imaging of the vessel wall of large murine arteries using two-photon laser scanning microscopy (TPLSM).
    No preview · Article · Jan 2008
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    ABSTRACT: We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E(-/-) mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E(-/-) mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.
    Full-text · Article · Jul 2007 · Molecular Imaging
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    ABSTRACT: This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell-vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.
    Full-text · Article · Jul 2007 · Pflügers Archiv - European Journal of Physiology
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    ABSTRACT: Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.
    No preview · Article · May 2007 · Microscopy Research and Technique
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    ABSTRACT: A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.
    Full-text · Article · Feb 2007 · Nano Letters
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    ABSTRACT: Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 microm) and density (0.045 vs. 0.57 microm(-2)) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 microm(-3)), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 microm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.
    Full-text · Article · Feb 2007 · Journal of Vascular Research
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    ABSTRACT: Microvascular surgery for the reconstruction of complex defects involves an ischemic period, which may cause flap failure as the result of ischemia/reperfusion injury. We assessed the microvascular consequences of rat cremaster muscle transplantation after prolonged periods of cold storage in HTK-Bretschneider solution (HTK). Cremaster muscle transplantations were performed immediately or after 8 or 24 h of cold storage (4 degrees C) in HTK or saline. Intravital microscopy was used to quantify capillary perfusion and venular leukocyte-endothelium interactions following transplantation. The transplantation procedure itself resulted in 50-65 min of ischemia. After direct transplantation, capillary perfusion was 90% of control. Transplantation after 8 h of cold storage in either HTK or saline did not deteriorate capillary perfusion. When the tissue was stored for 24 h, HTK was superior to saline in preserving capillary perfusion (HTK: 76-83% of control, saline: 30%). Immediate transplantation induced a small increase in leukocyte adhesion. Prolonged cold storage in either fluid resulted in reduced flow velocities (qualitative observations) and edema formation, which hampered quantification of leukocyte-endothelium interactions. Even after 8 or 24 h of cold storage in HTK, transplantation of rat cremaster muscle was successful with good capillary perfusion. Capillary perfusion was better preserved in HTK than in saline.
    Full-text · Article · Apr 2006 · Journal of Surgical Research
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    ABSTRACT: Hypothermia is an important preservation method for tissues and solid organs. The aim of the present study was to assess in rat cremaster muscle the effect of hypothermia, without or with pre-ischaemic HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution) perfusion, on microvascular consequences of 4 or 6 h ischaemia and 2 h of reperfusion. Intravital microscopy was applied to examine capillary perfusion and leucocyte-endothelium interactions. The cremaster muscle was subjected to 4 or 6 h of cold (4 degrees C) or warm (33-34 degrees C) ischaemia and 2 h of reperfusion. Measurements were performed at baseline, prior to HTK perfusion and ischaemia, and at 0, 1 and 2 h after blood flow restoration. Hypothermia completely prevented the 50% reduction in capillary perfusion that was observed previously at start of reperfusion after 4 h warm ischaemia. After 6 h of warm ischaemia, perfusion resumed in only 45% of capillaries and remained at this low level during reperfusion. In contrast, only a slight decrease (< 10%) in capillary perfusion was observed after 6 h of cold ischaemia. Pre-ischaemic HTK perfusion had no beneficial effect on tissue perfusion. Both hypothermia and HTK attenuated the significant increase in venular leucocyte-vessel wall interactions, which was observed after 4 h of warm ischaemia in a previous study. Combined application of both interventions had no additional effects. After 6 h of warm ischaemia, no increase in leucocyte-vessel wall interactions was observed, possibly due to venular flow reduction. In conclusion, hypothermia preserves capillary perfusion and prevents an increase in leucocyte-vessel wall interactions during reperfusion after muscle tissue ischaemia. Preischaemic perfusion of the vasculature with HTK does not improve the effects of cold storage on tissue perfusion, but attenuates the inflammatory response independently of temperature effect.
    Full-text · Article · Aug 2005 · Clinical Science

Publication Stats

4k Citations
532.31 Total Impact Points


  • 1997-2011
    • Technische Universiteit Eindhoven
      • • Department of Biomedical Engineering
      • • Department of Mechanical Engineering
      Eindhoven, North Brabant, Netherlands
  • 1982-2009
    • Maastricht University
      • • Department of Physiology
      • • Department of Pharmacology
      Maestricht, Limburg, Netherlands
  • 2001
    • Utrecht University
      Utrecht, Utrecht, Netherlands
  • 1993-2001
    • Maastricht Universitair Medisch Centrum
      • Central Diagnostic Laboratory
      Maestricht, Limburg, Netherlands
  • 1995
    • Academisch Medisch Centrum Universiteit van Amsterdam
      • Department of Surgery
      Amsterdamo, North Holland, Netherlands
  • 1983-1995
    • Transnationale Universiteit Limburg
      Box Elder, South Dakota, United States
  • 1991
    • University of Louisville
      Louisville, Kentucky, United States
  • 1987
    • University of Washington Seattle
      • Department of Physiology and Biophysics
      Seattle, WA, United States