Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been descri... more Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been described in developing vasculature of many organs and organisms. The aim of this study was to resolve the question about pillar formation and their further maturation employing zebrafish caudal vein plexus (CVP). The CVP development was monitored by in vivo confocal microscopy in high spatio-temporal resolution using the transgenic zebrafish model Fli1a:eGPF//Gata1:dsRed. We tracked back the formation of pillars (diameter ≤ 4 µm) and intercapillary meshes (diameter > 4 µm) and analysed their morphology and behaviour. Transluminal pillars in the CVP arose via a combination of sprouting, lumen expansion, and/or the creation of intraluminal folds, and those mechanisms were not associated directly with blood flow. The follow-up of pillars indicated that one-third of them disappeared between 28 and 48 h post fertilisation (hpf), and of the remaining ones, only 1/17 changed their cross-section a...
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin an... more Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterizat...
Nuclear emulsions have been widely used in particle physics to identify new particles through the... more Nuclear emulsions have been widely used in particle physics to identify new particles through the observation of their decays thanks to their unique spatial resolution. Nevertheless, before the advent of automatic scanning systems, the emulsion analysis was very demanding in terms of well trained manpower. Due to this reason, they were gradually replaced by electronic detectors, until the '90s, when automatic microscopes started to be developed in Japan and in Europe. Automatic scanning was essential to conceive large scale emulsion-based neutrino experiments like CHORUS, DONUT and OPERA. Standard scanning systems have been initially designed to recognize tracks within a limited angular acceptance (θ 30°) where θ is the track angle with respect to a line perpendicular to the emulsion plane. In this paper we describe the implementation of a novel fast automatic scanning system aimed at extending the track recognition to the full angular range and improving the present scanning speed. Indeed, nuclear emulsions do not have any intrinsic limit to detect particle direction. Such improvement opens new perspectives to use nuclear emulsions in several fields in addition to large scale neutrino experiments, like muon radiography, medical applications and dark matter directional detection.
The development of automatic scanning systems was a fundamental issue for large scale neutrino de... more The development of automatic scanning systems was a fundamental issue for large scale neutrino detectors exploiting nuclear emulsions as particle trackers. Such systems speed up significantly the event analysis in emulsion, allowing the feasibility of experiments with unprecedented statistics. In the early 1990s, R&D programs were carried out by Japanese and European laboratories leading to automatic scanning systems more and more efficient. The recent progress in the technology of digital signal processing and of image acquisition allows the fulfillment of new systems with higher performances. In this paper we report the description and the performance of a new generation scanning system able to operate at the record speed of 84 cm2/hour and based on the Large Angle Scanning System for OPERA (LASSO) software infrastructure developed by the Naples scanning group. Such improvement, reduces the scanning time by a factor 4 with respect to the available systems, allowing the readout of huge amount of nuclear emulsions in reasonable time. This opens new perspectives for the employment of such detectors in a wider variety of applications.
Meninges cover the surface of the brain and spinal cord and contribute to protection and immune s... more Meninges cover the surface of the brain and spinal cord and contribute to protection and immune surveillance of the central nervous system (CNS). How the meningeal layers establish CNS compartments with different accessibility to immune cells and immune mediators is, however, not well understood. Here, using 2-photon imaging in female transgenic reporter mice, we describe VE-cadherin at intercellular junctions of arachnoid and pia mater cells that form the leptomeninges and border the subarachnoid space (SAS) filled with cerebrospinal fluid (CSF). VE-cadherin expression also marked a layer of Prox1+ cells located within the arachnoid beneath and separate from E-cadherin+ arachnoid barrier cells. In vivo imaging of the spinal cord and brain in female VE-cadherin-GFP reporter mice allowed for direct observation of accessibility of CSF derived tracers and T cells into the SAS bordered by the arachnoid and pia mater during health and neuroinflammation, and detection of volume changes of...
The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the cent... more The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multi-step T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro.Here we present the Under-Flow Migration Tracker (UFMTrack) framework and a pipeline built based on it to study the entire multi-step extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro.UFMTrack a...
Understanding the meteorological drivers of extreme impacts in social or environmental systems is... more Understanding the meteorological drivers of extreme impacts in social or environmental systems is important to better quantify current and project future climate risks. Impacts are typically an aggregated response to many different interacting drivers at various temporal scales, rendering such driver identification a challenging task. Machine learning–based approaches, such as deep neural networks, may be able to address this task but require large training datasets. Here, we explore the ability of Convolutional Neural Networks (CNNs) to predict years with extremely low gross primary production (GPP) from daily weather data in three different vegetation types. To circumvent data limitations in observations, we simulate 100,000 years of daily weather with a weather generator for three different geographical sites and subsequently simulate vegetation dynamics with a complex vegetation model. For each resulting vegetation distribution, we then train two different CNNs to classify daily...
In recent years, muon radiography has been successfully applied to tackle geological issues and h... more In recent years, muon radiography has been successfully applied to tackle geological issues and has enjoyed an increasing interest, mainly because this methodology enriches the geophysical arsenal by another shallow subsurface imaging tool that may give independent constraints on material density. Muons that originate from the collision of cosmic particles with Earth’s atmosphere are able to penetrate the material in question and can finally be recorded by a detector. The irradiation intensity can then be inverted to the density of the traversed material. Various successful two-dimensional attempts have already been made to image e.g. magma chambers inside volcanoes (Lesparre et al., 2012; Nishiyama et al., 2014; Tanaka et al., 2005), but this method has yet to be applied for mapping the base of glaciers, where the density contrasts between ice and underlying bedrock are even greater than those between magma and host rock. While a high Alpine setup limits the possibilities to deploy...
Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space o... more Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery. After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.
The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, me... more The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, mesothelial and glial brain barriers, which strictly control the passage of molecules, solutes and immune cells. While the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) have been extensively investigated, less is known about the epithelial and mesothelial arachnoid barrier and the glia limitans. Here, we summarize current knowledge of the cellular composition of the brain barriers with a specific focus on describing the molecular constituents of their junctional complexes. We propose that the brain barriers maintain CNS immune privilege by dividing the CNS into compartments that differ with regard to their role in immune surveillance of the CNS. We close by providing a brief overview on experimental tools allowing for reliable in vivo visualization of the brain barriers and their junctional complexes and thus the respective CNS compartme...
T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (N... more T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b−/− CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b−/− CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b−/− CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surve...
Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been descri... more Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been described in developing vasculature of many organs and organisms. The aim of this study was to resolve the question about pillar formation and their further maturation employing zebrafish caudal vein plexus (CVP). The CVP development was monitored by in vivo confocal microscopy in high spatio-temporal resolution using the transgenic zebrafish model Fli1a:eGPF//Gata1:dsRed. We tracked back the formation of pillars (diameter ≤ 4 µm) and intercapillary meshes (diameter > 4 µm) and analysed their morphology and behaviour. Transluminal pillars in the CVP arose via a combination of sprouting, lumen expansion, and/or the creation of intraluminal folds, and those mechanisms were not associated directly with blood flow. The follow-up of pillars indicated that one-third of them disappeared between 28 and 48 h post fertilisation (hpf), and of the remaining ones, only 1/17 changed their cross-section a...
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin an... more Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterizat...
Nuclear emulsions have been widely used in particle physics to identify new particles through the... more Nuclear emulsions have been widely used in particle physics to identify new particles through the observation of their decays thanks to their unique spatial resolution. Nevertheless, before the advent of automatic scanning systems, the emulsion analysis was very demanding in terms of well trained manpower. Due to this reason, they were gradually replaced by electronic detectors, until the '90s, when automatic microscopes started to be developed in Japan and in Europe. Automatic scanning was essential to conceive large scale emulsion-based neutrino experiments like CHORUS, DONUT and OPERA. Standard scanning systems have been initially designed to recognize tracks within a limited angular acceptance (θ 30°) where θ is the track angle with respect to a line perpendicular to the emulsion plane. In this paper we describe the implementation of a novel fast automatic scanning system aimed at extending the track recognition to the full angular range and improving the present scanning speed. Indeed, nuclear emulsions do not have any intrinsic limit to detect particle direction. Such improvement opens new perspectives to use nuclear emulsions in several fields in addition to large scale neutrino experiments, like muon radiography, medical applications and dark matter directional detection.
The development of automatic scanning systems was a fundamental issue for large scale neutrino de... more The development of automatic scanning systems was a fundamental issue for large scale neutrino detectors exploiting nuclear emulsions as particle trackers. Such systems speed up significantly the event analysis in emulsion, allowing the feasibility of experiments with unprecedented statistics. In the early 1990s, R&D programs were carried out by Japanese and European laboratories leading to automatic scanning systems more and more efficient. The recent progress in the technology of digital signal processing and of image acquisition allows the fulfillment of new systems with higher performances. In this paper we report the description and the performance of a new generation scanning system able to operate at the record speed of 84 cm2/hour and based on the Large Angle Scanning System for OPERA (LASSO) software infrastructure developed by the Naples scanning group. Such improvement, reduces the scanning time by a factor 4 with respect to the available systems, allowing the readout of huge amount of nuclear emulsions in reasonable time. This opens new perspectives for the employment of such detectors in a wider variety of applications.
Meninges cover the surface of the brain and spinal cord and contribute to protection and immune s... more Meninges cover the surface of the brain and spinal cord and contribute to protection and immune surveillance of the central nervous system (CNS). How the meningeal layers establish CNS compartments with different accessibility to immune cells and immune mediators is, however, not well understood. Here, using 2-photon imaging in female transgenic reporter mice, we describe VE-cadherin at intercellular junctions of arachnoid and pia mater cells that form the leptomeninges and border the subarachnoid space (SAS) filled with cerebrospinal fluid (CSF). VE-cadherin expression also marked a layer of Prox1+ cells located within the arachnoid beneath and separate from E-cadherin+ arachnoid barrier cells. In vivo imaging of the spinal cord and brain in female VE-cadherin-GFP reporter mice allowed for direct observation of accessibility of CSF derived tracers and T cells into the SAS bordered by the arachnoid and pia mater during health and neuroinflammation, and detection of volume changes of...
The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the cent... more The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multi-step T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro.Here we present the Under-Flow Migration Tracker (UFMTrack) framework and a pipeline built based on it to study the entire multi-step extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro.UFMTrack a...
Understanding the meteorological drivers of extreme impacts in social or environmental systems is... more Understanding the meteorological drivers of extreme impacts in social or environmental systems is important to better quantify current and project future climate risks. Impacts are typically an aggregated response to many different interacting drivers at various temporal scales, rendering such driver identification a challenging task. Machine learning–based approaches, such as deep neural networks, may be able to address this task but require large training datasets. Here, we explore the ability of Convolutional Neural Networks (CNNs) to predict years with extremely low gross primary production (GPP) from daily weather data in three different vegetation types. To circumvent data limitations in observations, we simulate 100,000 years of daily weather with a weather generator for three different geographical sites and subsequently simulate vegetation dynamics with a complex vegetation model. For each resulting vegetation distribution, we then train two different CNNs to classify daily...
In recent years, muon radiography has been successfully applied to tackle geological issues and h... more In recent years, muon radiography has been successfully applied to tackle geological issues and has enjoyed an increasing interest, mainly because this methodology enriches the geophysical arsenal by another shallow subsurface imaging tool that may give independent constraints on material density. Muons that originate from the collision of cosmic particles with Earth’s atmosphere are able to penetrate the material in question and can finally be recorded by a detector. The irradiation intensity can then be inverted to the density of the traversed material. Various successful two-dimensional attempts have already been made to image e.g. magma chambers inside volcanoes (Lesparre et al., 2012; Nishiyama et al., 2014; Tanaka et al., 2005), but this method has yet to be applied for mapping the base of glaciers, where the density contrasts between ice and underlying bedrock are even greater than those between magma and host rock. While a high Alpine setup limits the possibilities to deploy...
Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space o... more Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery. After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.
The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, me... more The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, mesothelial and glial brain barriers, which strictly control the passage of molecules, solutes and immune cells. While the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) have been extensively investigated, less is known about the epithelial and mesothelial arachnoid barrier and the glia limitans. Here, we summarize current knowledge of the cellular composition of the brain barriers with a specific focus on describing the molecular constituents of their junctional complexes. We propose that the brain barriers maintain CNS immune privilege by dividing the CNS into compartments that differ with regard to their role in immune surveillance of the CNS. We close by providing a brief overview on experimental tools allowing for reliable in vivo visualization of the brain barriers and their junctional complexes and thus the respective CNS compartme...
T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (N... more T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b−/− CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b−/− CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b−/− CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surve...
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Papers by Mykhailo Vladymyrov