Enhancement and Segmentation Workflow for the Developing Zebrafish Vasculature †

Zebrafish have become an established in vivo vertebrate model to study cardiovascular development and disease. However, most published studies of the zebrafish vascular architecture rely on subjective visual assessment, rather than objective quantification. In this paper, we used state-of-the-art light sheet fluorescence microscopy to visualize the vasculature in transgenic fluorescent reporter zebrafish. Analysis of image quality, vascular enhancement methods, and segmentation approaches were performed in the framework of the open-source software Fiji to allow dissemination and reproducibility. Here, we build on a previously developed image processing pipeline; evaluate its applicability to a wider range of data; apply and evaluate an alternative vascular enhancement method; and, finally, suggest a work-flow for successful segmentation of the embryonic zebrafish vasculature

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and EC number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and EC number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds

Cerebrovascular development: mechanisms and experimental approaches

The cerebral vasculature plays a central role in human health and disease and possesses several unique anatomic, functional and molecular characteristics. Despite their importance, the mechanisms that determine cerebrovascular development are less well studied than other vascular territories. This is in part due to limitations of existing models and techniques for visualisation and manipulation of the cerebral vasculature. In this review we summarise the experimental approaches used to study the cerebral vessels and the mechanisms that contribute to their development

Cerebrovascular development: mechanisms and experimental approaches

The cerebral vasculature plays a central role in human health and disease and possesses several unique anatomic, functional and molecular characteristics. Despite their importance, the mechanisms that determine cerebrovascular development are less well studied than other vascular territories. This is in part due to limitations of existing models and techniques for visualisation and manipulation of the cerebral vasculature. In this review we summarise the experimental approaches used to study the cerebral vessels and the mechanisms that contribute to their development

3D Quantification and Description of the Developing Zebrafish Cranial Vasculature

Background: Zebrafish are an excellent model to study cardiovascular development and disease. Transgenic reporter lines and state-of-the-art microscopy allow 3D visualization of the vasculature in vivo. Previous studies relied on subjective visual interpretation of vascular topology without objective quantification. Thus, there is the need to develop analysis approaches that model and quantify the zebrafish vasculature to understand the effect of development, genetic manipulation or drug treatment. Aim: To establish an image analysis pipeline to extract quantitative 3D parameters describing the shape and topology of the zebrafish vasculature, and examine how these are impacted during development, disease, and by chemicals. Methods: Experiments were performed in zebrafish embryos, conforming with UK Home Office regulations. Image acquisition of transgenic zebrafish was performed using a Z.1 Zeiss light-sheet fluorescence microscope. Pre-processing, enhancement, registration, segmentation, and quantification methods were developed and optimised using open-source software, Fiji (Fiji 1.51p; National Institutes of Health, Bethesda, USA). Results: Motion correction was successfully applied using Scale Invariant Feature Transform (SIFT), and vascular enhancement based on vessel tubularity (Sato filter) exceeded general filter outcomes. Following evaluation and optimisation of a variety of segmentation methods, intensity-based segmentation (Otsu thresholding) was found to deliver the most reliable segmentation, allowing 3D vascular volume measurement. Following successful segmentation of the cerebral vasculature, a workflow to quantify left-right intra-sample symmetry was developed, finding no difference from 2-to-5dpf. Next, the first vascular inter-sample registration using a manual landmark-based approach was developed and it was found that conjugate direction search allowed automatic inter-sample registration. This enabled extraction of age-specific regions of similarity and variability between different individual embryos from 2-to-5dpf. A workflow was developed to quantify vascular network length, branching points, diameter, and complexity, showing reductions in zebrafish without blood flow. Also, I discovered and characterised a previously undescribed endothelial cell membrane behaviour termed kugeln. Conclusion: A workflow that successfully extracts the zebrafish vasculature and enables detailed quantification of a wide variety of vascular parameters was developed

Development and characterisation of a zebrafish larval model to investigate mechanisms for pathophysiology of intracranial hypertension in cryptococcal meningitis

Cryptococcosis is a fungal infection caused by members of the genus Cryptococcus. Worldwide, the most prevalent pathogen of this genus is the encapsulated saprophyte species Cryptococcus neoformans. Cryptococcosis most commonly occurs as an opportunistic airborne lung infection which can disseminate to most organ systems. The central nervous system appears particularly susceptible to developing a pathology from the infection, with more than half of cryptococcosis patients diagnosed with cryptococcal meningitis despite infection in multiple organs. Cryptococcal meningitis (CM) is a meningoencephalitis (infection of the brain parenchyma and meninges) which globally accounts for 19% (13-24) of AIDS-related mortality (Rajansingham et al., 2022). In 2020, reports show annual incidence of 152 000 cases of cryptococcal meningitis, resulting in 112 000 cryptococcal-related deaths, almost half of which are in eastern and southern Africa (Rajansingham et al., 2022). 50-70% of CM cases present with a pathologically elevated intracranial pressure (intracranial hypertension) (Graybill et al., 2000; Jarvis et al., 2014; Kagimu et al., 2022;). This thesis aims to improve our understanding of intracranial hypertension in CM to help identify potential targets for treatment, by developing and testing new models of intracranial hypertension in CM. Three different approaches were used – theoretical, in vitro rheology and in vivo in zebrafish. Zebrafish was chosen as the core experimental system in which to develop new models due to its physiology, tractability for live imaging and susceptibility to cryptococcosis. In zebrafish larvae, the dynamic nature of cranial vasculature compartments and the CSF during infection was examined using wide field and light sheet microscopy techniques. The physical properties of tissues and fluids when interacting with cryptococcal yeast cells was modelled with theoretical and in vitro rheological measurements. In vitro it was found that viscosity of fluids may increase in the presence of heat killed C. neoformans, but whether this change is pathologically significant requires further investigation. Using a model of cryptococcal infection in zebrafish larvae, a “pulsation” phenomenon was identified, consisting of vasodilation and constriction in the cranial vasculature with an impact on vessel wall permeability. The findings in this work, are reflective of the CM pathology as seen in human patients and suggest impaired CSF and blood flow homeostasis may contribute to intracranial hypertension in CM