Albumin uptake and distribution in the zebrafish liver as observed via correlative imaging
Introduction
The liver sinusoidal unit is comprised of parenchymal cells (hepatocytes) and non-parenchymal cells (liver sinusoidal cells) [9]. Together, they form a structural and functional micro-anatomical unit thereby tightly controlling various essential liver functions, including liver homeostasis [51]. Most importantly, the liver health is largely dependent upon the structural and functional intactness of the liver sinusoidal unit [71]. Briefly, the blood enters the liver via the portal tracts and passes through the sinusoidal vasculature (i.e., sinusoids) that is delineated by the liver sinusoidal endothelial cells (LSECs) to finally leave the liver circulation via the central vein. Located between the sinusoidal endothelial lining and the hepatocytes, the space of Disse is largely occupied by the microvillus projections of the hepatocytes.
Hepatic transport of macromolecules (e.g., albumin, lipoproteins and waste products) is normally confined around the liver sinusoids. LSECs play a major role in the process of tightly regulating transport between the sinusoidal blood and the surrounding liver tissue. Transport mechanisms involve endocytosis-mediated intracellular transport [54] and size-dependent trans-endothelial filtration [6]. For this, LSECs are equipped with unique structural and functional features for the processing and transport of substances and solutes. LSECs possess an extensive endolysosomal apparatus, characterised by the abundance of bristle-coated vesicles, primary and secondary lysosomes [68], [69]. Previous studies have shown that LSECs are one of the most effective scavenger cells in clearing waste products, proteins and lipids from the circulation [21]. Besides the presence of a well-developed endocytic system, LSECs bear open pores lacking a diaphragm—known as fenestrae or fenestrations—allowing the free bi-directional exchange of particles between the sinusoidal blood, the space of Disse and the microvillus surface of hepatocytes [72]. As a whole, LSECs act as a gatekeeper within the liver sinusoidal unit, selectively controlling the passage of sinusoidal blood constituents into the deeper laying parenchymal cells of the liver [48].
Liver transport around the hepatic sinusoids has been extensively studied. The majority of in vivo and in vitro studies utilise rodents as an experimental animal model to research liver biology and pathobiology. These rodent models have proven largely beneficial for the study of cancer, fibrosis and cellular transport to name a few [38]. Often these studies provided a direct translational impact to pre-clinical relevant settings [10]. One technical challenge is the ability to probe large-volume hepatic functions within in vivo and in situ settings in rodent studies. This is mainly due to the limiting optical transparency and size of the animals, making high-throughput observations and consistent large-volume assessments via microscopy difficult. The latter is particularly relevant in the observation of rare events such as the onset of cancer, or when sporadic transport processes need to be recorded. The zebrafish experimental model possesses some advantages to overcome these limitations. In cell and drug-based transport studies, zebrafish have already been successfully employed to study cell metastasis [29], immune responses [43], [58], and to monitor nanoparticle transport within different organs [12], [26]. In addition, zebrafish physiological and pathophysiological studies have demonstrated a strong translational aspect to human physiology [47], [62].
In this study, we aimed to rigorously define the microarchitecture of the zebrafish liver sinusoidal unit at the cell-tissue level, and critically compare the zebrafish liver ultrastructure to the existing rodent literature. We further assessed cellular transport routes in the zebrafish using the well-established experimental model of albumin liver transport [34], [63]. To support our structure-function studies, we utilised contemporary correlative light and electron microscopy (CLEM) approaches, including scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tomography (TET) [32], [36], [57]. Based on our cross-correlative results, we propose the zebrafish liver as a viable alternative experimental model for hepatic transport studies. This statement is underpinned by our multimodal and correlated imaging studies, which allowed the quantification on the hepatic distribution of macromolecular complexes of interest.
Section snippets
Animal model
According to standard protocols, zebrafish (Danio rerio) were housed in a purpose-built zebrafish facility at Macquarie University (Australia) and experiments were conducted under Macquarie University Animal Ethics and Biosafety approvals (2012/050 and 2015/033; 5201401007). Casper zebrafish lines (nacre:roy orbison) were raised and maintained in clear system water at 28 °C in a 13 h light and 11 h dark cycle and embryos were collected by natural spawning and raised at 28.5 °C in E3 solution
Correlative 3D microscopic mapping of the zebrafish hepatic microarchitecture
To study the ultrastructural characteristics of zebrafish liver in 2D and 3D, we processed zebrafish larvae for electron microscopy and imaged sections of the liver using TEM and TET. These high-resolution imaging techniques revealed similarities in the hepatic cellular organisation compared to rodents and humans. For example, we confirmed the presence of fine ultrastructural features such as glycogen, lipid droplets and lysosomes in addition to the existence of the commonly found organelles
Discussion
The transport pathways along the hepatic sinusoids have been studied in rodents and human for many decades [18], [45], [73]. It has been well established that any material (solutes, particles, proteins and lipids) that end up within the space of Disse and subsequently, the hepatocytes, is tightly controlled by the LSECs by means of fenestrae-mediated sieving or clathrin-mediated transcytosis. Earlier work on rodents focused on lipoprotein transport in the liver [3], [44] and the
Conclusion
The zebrafish liver sinusoidal unit displays a unique structural resemblance compared to the humans and rodents’ counterparts. Likewise, functional studies revealed comparable macromolecular transport routes for albumin, including subcellular processing pathways, highlighting the viability of the zebrafish liver sinusoidal unit forward as an attractive experimental alternative to study liver biology and pathology.
Acknowledgements
The authors acknowledge the facilities as well as technical assistance from staff of the Microscopy Australia node - the Australian Centre for Microscopy & Microanalysis - at The University of Sydney, as well as the Zebrafish Facility at Macquarie University. F.B. is indebted to the Australian Research Council (ARC) for funding some of the work reported herein (ARC LE110100203).
Competing interest
The authors declare no competing interest.
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