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

Elisabeth Kugler, Ryan Snodgrass, George Bowley, Karen Plant, Jovana Serbanovic-Canic, Paul C. Evans, Timothy Chico, Paul Armitage

Posted on: 3 September 2020

Preprint posted on 24 July 2020

Article now published in Vascular Biology at http://dx.doi.org/10.1530/vb-21-0009

Microscopy and zebrafish: Understanding blood-flow and its effects on different vascular beds – part I.

Selected by Mariana De Niz

Categories: cell biology, developmental biology

Background

Endothelial cells perform crucial functions in health and disease. They are important for physiological processes including wound healing, tissue regeneration, immune responses, menstruation and pregnancy. Increasing evidence suggests endothelial cells display different molecular and functional properties according to their anatomical site, emphasising the need to study endothelial cell function in different vascular beds. Zebrafish are a conventionally used model organism to study the vasculature in health and disease.|Zebrafish embryonic transparency allows non-invasive studies of different vascular beds in the same animal, and zebrafish embryos are well-established models to study the role of blood flow on vasculogenesis and angiogenesis.

Important questions that remain unanswered about the role of blood flow in vascular development include whether the absence of blood flow induces similar effects in different vascular beds, and what that effect is over time. In their work, Kugler et al performed LSFM 3D in vivo imaging to study the head and trunk vasculature of zebrafish embryos with and without blood flow (1).

Figure 1. Rows 1-2: The effect of tnnt2a morpholino knockdown on cerebral vessel development. Rows 3-4: The effect of absent blood flow on trunk vessel development. (From Ref 1).

Key findings and developments

The study by Kugler et al begins by determining whether cerebrovascular patterning is impaired by absent blood flow. The authors began by comparing the cerebral vascular morphology of controls, control morpholinos, and troponin T2A morpholinos that have inhibited development of cardiac contraction and impeded blood flow. The latter displayed abnormal cerebral vasculature, with vessels in the midbrain severely affected, and an enlarged primary head sinus, while the perineural vessels remained relatively normal. Examination of the distribution of endothelial cells and nuclear density in these animals supported these findings. Altogether, the main finding was that the effects of lack of blood flow become more severe over time, and that central vessels are more impacted than those in the periphery.

The study continued by examining vascular patterning and endothelial cell nuclei in the trunk of the 3 groups of animals previously established. In contrast to cerebral vessels, overall, trunk vessel patterning seemed unaltered in the absence of blood flow, except for the cardinal vein which seemed affected by lack of blood flow as remodelling, including intussusception – the creation of new blood vessels created by splitting of an existing blood vessel into two, seemed lacking confirming previous findings by (Karthik et al., 2018 (2)).

The authors went on to elucidate the effect of absent blood flow over time by quantifying cerebrovascular area and number. They found a significant reduction in vascular area and endothelial cell number in troponin T2A morphants. During development, the number of nuclei-to-vasculature increased in controls, but decreased in animals without blood flow. A similar analysis in the trunk showed that the vascular area was significantly reduced by absent blood flow early in embryo development, but not later. Altogether, these results suggested that the cerebral vessels were more severely affected than the trunk vessels.

To determine whether vessels of different identity were differentially impacted by lack of blood flow, the diameter of different veins and arteries in the brain, trunk and tail were quantified. Cerebral arterial diameter was reduced by 2.5% in troponin T2A morphants compared to controls, while posterior cerebral vein diameter was reduced by 36%. In the trunk, the mean arterial diameter was reduced by 40%, while the venous diameter was reduced by 46%. Finally, the dorsal aorta was reduced by 41%, while the posterior cardinal vein was reduced by 29%. These results suggest that vessels of arterial and venous identity are both affected by lack of blood flow, but specific differences exist in various anatomic locations.

The study then went on to explore whether cell death contributed to a reduction in endothelial cell numbers in the troponin T2A morphants. In the head and trunk, cell death appeared to be increased in the morphants, however more detailed analysis suggested the increased signal of the cell death marker came from non-vascular foci, showing that increased cell death occurs in the absence of blood flow, but is not endothelial-cell specific.

The authors hypothesized that perhaps the increased cell death in the troponin T2A morphants might alter the inflammatory response present in different anatomical locations. However, no difference was found in the number of neutrophils or macrophages between the three zebrafish groups used in the study. Nitric oxide, which was used as indirect inflammation read-out, also did not appear to differ in the head or trunk. Altogether, the data suggest that the lack of blood flow increases cell death, but not tissue inflammation.

What I like about this preprint

I find vascular biology very interesting and I think the authors used an exciting imaging method and an exciting model to answer questions on vascular biology, relevant to various field of research.

References

Kugler et al, The effect of absent blood flow on the zebrafish cerebral and trunk vasculature, bioRxiv, 2020.
Karthik, S., et al., Synergistic interaction of sprouting and intussusceptive angiogenesis during zebrafish caudal vein plexus development. Scientific Reports 8, 2018.

doi: https://doi.org/10.1242/prelights.24413

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Author's response

Elisabeth Kugler, Timothy Chico, Paul Armitage shared

Open questions

1. Do you expect the effects of lack of blood flow in adult zebrafish to be similar to those observed in embryos? Is it possible to do conditional knock-downs to explore this?

In this study we examined how lacking blood flow impacts very early vascular development, rather than what happens when blood flow is lacking after the vasculature is established. Studying the role of lacking blood flow in adults is technically challenging as their survival depends on heartbeat, while embryos can survive up to 5-7 days post fertilization without blood flow. However, we would assume that inhibiting blood flow in adults has certain parallels to what we observe in embryos, namely: (a) changes in vessel diameters, (b) reduced endothelial cell survival, and (c) that cerebral vessels are potentially also more severely affected. In contrast, as heart contraction can only be stopped temporarily in adults, we would assume that vascular patterning impacts would be less pronounced or completely lacking.

2.Besides the differences between the brain and trunk, do you know if other organs in the zebrafish also show effects due to impeded blood flow? Moreover, have the effects of lack of blood flow been studied in other animal models or humans, and are these consistent with your findings?

Due to the importance of blood flow in health and disease, many studies exist studying its effects on particular organs or sub-regions. As zebrafish are an excellent model to study the role blood flow, due to their transparency and ability to survive for a few days blood flow was shown to play a role in swimbladder vasculogenesis, cardiac trabeculation and valvulogenesis, suppresses Cerebral Cavernous Malformations (CCMs), and effect endothelial cell extrusions (Winata et al., 2010; Banjo et al., 2013; Campinho et al., 2020; Rödel et al., 2019), just to name a few. More work is needed to really understand in detail the molecular and cellular mechanisms that are regulated by blood flow, with our study adding some information on the role of blood flow over time and comparing different vascular beds for the first time. Notably, the physiology of endothelial cells under no flow in zebrafish embryos also shows similarities to the behaviour of cells under disease-causing disturbed blood flow in humans and mice (e.g. Serbanovic-Canic et al 2017), suggesting that zebrafish embryos can be a useful model to study vascular disease processes.

3.Can you differentiate cell death modes due to lack of blood flow? Among your findings, you describe that increased cell death upon lack of blood flow is not specific to endothelial cells. Do you know if it is specific to any cell type or anatomical structure?

There are several forms of cell death, including apoptosis, necrosis, and autophagy. Future work is needed for us to identify which form of cell death(s) we observe. Understanding the form(s) of cell death and their temporal patterns might then also help us understand which cell types or anatomical structures are particularly effected. Previous studies from our group found increased endothelial cell apoptosis under no flow conditions (e.g. Serbanovic-Canic et al 2017) but more work is needed to evaluate other mechanisms of cell death.

4.Have you studied the effects of impeded blood flow due to pathology – for instance, infections or diseases that alter blood flow? What would your hypothesis be for such a study?

Our groups involved in this project come from very diverse backgrounds, but one common theme is wanting to understand blood flow in pathology. This is exemplified by our studies on the impact of glucose on zebrafish tectal blood vessel patterning and neurovascular coupling (Chhabria et al., 2018), Notch signalling regulation by blood flow in zebrafish (Watson et al., 2013), shear stress-dependent anti-inflammatory mechanisms in mice (Le et al., 2016), establishing functional screens of flow-responsive genes in zebrafish (Serbanovic-Canic et al., 2016), and studying human cerebral small vessel diseases (Heye et al., 2016), ischemic stroke, and blood-brain-barrier permeability.

5.What would you expect to observe from intermittent disruption of blood flow? Or from selective blockage of specific vessel types but not others?

As briefly mentioned above, we would assume that a temporary disruption does not lead to the severe patterning effects we observe using tnnt2a morphants. However, it is likely that vessel diameters change as direct effect of lacking blood flow and that cell death increases.

Using a combination of pre-clinical models, future work might untangle the impact of hypoxia in this as zebrafish allow the examination of lacking blood flow without necessarily inducing hypoxia as it would be the case in mammalian models (Chico et al., 2008).

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

Background

Key findings and developments

What I like about this preprint

References

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