Platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy

Growing evidence supports the substantial pathophysiological impact of platelets on the development of acute lung injury. Methods for studying these cellular mechanisms in vivo are not present yet. The aim of this study was to develop a model enabling the quantitative analysis of platelet kinetics and platelet-endothelium interaction within consecutive segments of the pulmonary microcirculation in vivo. New Zealand White rabbits were anesthetized and ventilated. Autologous platelets were separated from blood and labeled ex vivo with rhodamine 6G. After implantation of a thoracic window, microhemodynamics and kinetics of platelets were investigated by intravital microscopy. Velocities of red blood cells (RBCs) and platelets were measured in arterioles, capillaries and venules, and the number of platelets adhering to the microvascular endothelium was counted. Kinetics of unstimulated platelets was compared with kinetics of thrombin-activated platelets. Velocity of unstimulated platelets was comparable to RBC velocity in all vessel segments. Unstimulated platelets passed the pulmonary microcirculation without substantial platelet-endothelial interaction. In contrast, velocity of activated platelets was decreased in all vascular segments indicating platelet margination and temporal platelet-endothelium interaction. Thrombin-activated platelets adhered to arteriolar endothelium; in capillaries and venules adherence of platelets was increased 8-fold and 13-fold, respectively. In conclusion, using intravital microscopy platelet kinetics were directly analyzed in the pulmonary microcirculation in vivo for the first time. In contrast to leukocytes, no substantial platelet-endothelium interaction occurs in the pulmonary microcirculation without any further stimulus. In response to platelet activation, molecular mechanisms enable adhesion of platelets in arterioles and venules as well as retention of platelets within capillaries. Copyright (C) 2002 S. Karger AG, Basel

Whole-brain vasculature reconstruction at the single capillary level

The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network

Ex vivo Dynamics of Human Glioblastoma Cells in a Microvasculature-on-a-Chip System Correlates with Tumor Heterogeneity and Subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Here, a microvasculature-on-a-chip system as a PVN model is used to evaluate the ex vivo dynamics of BTSCs from ten glioblastoma patients. BTSCs are found to preferentially localize in the perivascular zone, where they exhibit either the lowest motility, as in quiescent cells, or the highest motility, as in the invasive phenotype, with migration over long distance. These results indicate that PVN is a niche for BTSCs, while the microvascular tracks may serve as a path for tumor cell migration. The degree of colocalization between tumor cells and microvessels varies significantly across patients. To validate these results, single-cell transcriptome sequencing (10 patients and 21 750 single cells in total) is performed to identify tumor cell subtypes. The colocalization coefficient is found to positively correlate with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, a gene signature profile including PDGFRA correlates strongly with the “homing” of tumor cells to the PVN. These findings demonstrate that the model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient-specific tumor cell functions

Visualizing Structures in Confocal Microscopy Datasets Through Clusterization: A Case Study on Bile Ducts

Aiming at a better result from previous works, we employed some heuristics found in the literature to determine the appropriate parameters for the clustering. We proposed our methodology by adding some steps to be performed before the clustering phase: one step for pre-processing the volumetric dataset and another to analyzing candidate features to guide the clustering. In this latter aspect, we provide an interesting contribution: we have explored the gradient magnitude as a feature that allowed to extract relevant information from the density-based spatial clustering. Besides the fact that DBSCAN allows easy detection of noise points, an interesting result for both datasets was that the first and largest cluster found as significant for the visualization represents the structure of interest. In the red channel, this cluster represents the most prominent vessels, while in the green channel, the peribiliary glands were made more evident.Abstract—Three-dimensional datasets from biological tissues have increased with the evolution of confocal microscopy. Hepatology researchers have used confocal microscopy for investigating the microanatomy of bile ducts. Bile ducts are complex tubular tissues consisting of many juxtaposed microstructures with distinct characteristics. Since confocal images are difficult to segment because of the noise introduced during the specimen preparation, traditional quantitative analyses used in medical datasets are difficult to perform on confocal microscopy data and require extensive user intervention. Thus, the visual exploration and analysis of bile ducts pose a challenge in hepatology research, requiring different methods. This paper investigates the application of unsupervised machine learning to extract relevant structures from confocal microscopy datasets representing bile ducts. Our approach consists of pre-processing, clustering, and 3D visualization. For clustering, we explore the density-based spatial clustering for applications with noise (DBSCAN) algorithm, using gradient information for guiding the clustering. We obtained a better visualization of the most prominent vessels and internal structures.info:eu-repo/semantics/publishedVersio

Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood–brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo

Doctor of Philosophy

dissertationThis body of work aims at establishing an approach for intraoperative discrimination of cardiac tissue types based on fiber-optics confocal microscopy (FCM). An important application of this approach is in pediatric heart surgery. A major risk of these surgeries is surgically-induced trauma to the specialized tissue of the cardiac conduction system. Current clinical practice during pediatric heart surgery is to approximate the disposition of the conduction system and scrupulously avoid it. A method for real-time delineation of the conduction system during pediatric heart surgery is needed. FCM allows for real-time imaging of cellular and sub-cellular features up to 100 micrometers below a specimens surface. We hypothesized that an approach based on FCM and fluorescent extracellular dyes would allow for delineation of the conduction system. We investigated this approach in the living arrested heart of rodent. In addition, the approach was validated in fixed tissue preparations from rodent, hearts using immunohistochemistry, three-dimensional conventional confocal microscopy, and image processing. Furthermore, we investigated dye delivery and microdosing approaches for intraoperative FCM discrimination of cardiac tissue types motivated by concerns in regards to consumption and adverse reactions of the dyes used in clinical applications of FCM. Lastly, we assessed the performance of the human and automated classification systems in identifying cardiac tissue types acquired using this novel approach. We demonstrated that it was feasible to discriminate cardiac tissue types using FCM and extracellular dyes. In our investigation into dye delivery and microdosing approaches, we showed that the developed novel local dye delivery approach based on a foam agarose dye carrier is particularly suitable for FCM during pediatric heart surgery. Furthermore, both human and automated classification systems achieved similarly high sensitivity and specificity in discriminating cardiac tissue types including tissue of the conduction system. We suggest that this work constitutes an important step in clinical translation of FCM for cardiac tissue discrimination. The imaging approach as well as the foam agarose carrier for microdosed delivery of dye and the automated methods for tissue classification have the potential to reduce the incidence of trauma to the conduction system during pediatric heart surgery

Characterization of the Growth Hormone Secretagogue Receptor in Dilated Cardiomyopathy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease of skeletal and myocardial degeneration. Eventually, dilated cardiomyopathy develops from ischemia, inflammation and fibrosis. Due to the high mortality rate, there is an emerging need to diagnose DMD cardiomyopathy at early stages. Currently, DMD cardiomyopathy is diagnosed by imaging investigations and detection of circulating biomarkers. However, current imaging strategies detect functional and morphological changes but fall short in detecting molecular changes that underlie this disease. Circulating biomarkers provide information on the molecular level, but they are not cardiac-specific. Therefore, there is an emerging need for a biomarker that is endogenous to cardiac tissues. The growth hormone secretagogue receptor (GHSR) and its ligand, ghrelin are produced by both cardiomyocytes and vascular endothelial cells and could be an indicator of DMD cardiomyopathy. The work described in this thesis sought to characterize GHSR as a cardiac-localized biomarker in DMD cardiomyopathy. Histopathology and confocal imaging using a novel fluorescent ghrelin analog, Cy5-ghrelin(1-19), were used to investigate changes in cardiac tissue architecture and GHSR and inflammatory markers in the mdx:utrn-/- mouse model of DMD. My studies show that GHSR is elevated in mdx:utrn-/- myocardial tissues and correlate strongly with the macrophage marker F4-80 and the pro-inflammatory cytokine IL-6. Interestingly, I also show that both ghrelin and des-acyl ghrelin bind to sites in large cardiac vessels of mdx:utrn-/- which might be an indicator of vascular inflammation. Finally, my project shows the first report of GHSR in cardiac macrophages. In summary, my work suggests that, in dilated cardiomyopathy, elevations in GHSR correlate with the inflammatory phenotype as mediated by both the myocardium and macrophages

Fluorescence microscopy tensor imaging representations for large-scale dataset analysis

Understanding complex biological systems requires the system-wide characterization of cellular and molecular features. Recent advances in optical imaging technologies and chemical tissue clearing have facilitated the acquisition of whole-organ imaging datasets, but automated tools for their quantitative analysis and visualization are still lacking. We have here developed a visualization technique capable of providing whole-organ tensor imaging representations of local regional descriptors based on fluorescence data acquisition. This method enables rapid, multiscale, analysis and virtualization of large-volume, high-resolution complex biological data while generating 3D tractographic representations. Using the murine heart as a model, our method allowed us to analyze and interrogate the cardiac microvasculature and the tissue resident macrophage distribution and better infer and delineate the underlying structural network in unprecedented detail

Direct regional microvascular monitoring and assessment of blood brain barrier function following cerebral ischemia-reperfusion injury

Evans Blue (EB) is often used to evaluate Blood-Brain Barrier Damage (BBB) in cerebral ischemia, frequently by dye extraction. Herein we present a method that allows assessing regional brain microvasculature, distribution of EB and Fluorescent Isothiocyanate-Labeled Red Blood Cells (FITC-RBCs) in a rat model of acute cerebral Ischemia-Reperfusion (I-R). Wistar rats were subjected to 3 h of middle cerebral artery occlusion and then reperfused. At ~2.5 h of reperfusion, BBB opening was assessed by contrast enhanced magnetic resonance imaging. It was followed by injections of EB and FITC-RBCs that circulated for either 5 or 20 min. Regional microvasculature and tracer distributions were assessed by laser scanning confocal microscopy. Microvascular networks in stroke-affected regions networks were partially damaged with apparent EB extravasation. Brain regions were affected in the following order: preoptic area (PoA)\u3estriatum (Str)\u3ecortex (Ctx). EB leakage increased with circulation time in Str. Cells around the leakage sites sequestered EB. An inverse correlation was observed between low CBF rates recorded during MCA occlusion and post-reperfusion EB extravasation patterns. Accordingly, this approach provided data on brain regional microvascular status, extravascular tracer distribution and its cellular uptake. It may be useful to evaluate model-dependent variations in vascular injury and efficacy of putative vascular protective drugs in stroke

3-D Ultrasound Localization Microscopy for Identifying Microvascular Morphology Features of Tumor Angiogenesis at a Resolution Beyond the Diffraction Limit of Conventional Ultrasound

Angiogenesis has been known as a hallmark of solid tumor cancers for decades, yet ultrasound has been limited in its ability to detect the microvascular changes associated with malignancy. Here, we demonstrate the potential of 'ultrasound localization microscopy' applied volumetrically in combination with quantitative analysis of microvascular morphology, as an approach to overcome this limitation. This pilot study demonstrates our ability to image complex microvascular patterns associated with tumor angiogenesis in-vivo at a resolution of tens of microns - substantially better than the diffraction limit of traditional clinical ultrasound, yet using an 8 MHz clinical ultrasound probe. Furthermore, it is observed that data from healthy and tumor-bearing tissue exhibit significant differences in microvascular pattern and density. Results suggests that with continued development of these novel technologies, ultrasound has the potential to detect biomarkers of cancer based on the microvascular 'fingerprint' of malignant angiogenesis rather than through imaging of blood flow dynamics or the tumor mass itself