Using zebrafish larval models to study brain injury, locomotor and neuroinflammatory outcomes following intracerebral haemorrhage

Abstract

Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology.

Cerebrovascular development: mechanisms and experimental approaches

“…In intracerebral haemorrhage (ICH), macrophages play a role in the secondary post-stroke phase and represent a therapeutic target [304,305]. While mice have been the main model of choice to examine cerebral haemorrhage, zebrafish models also allow study of ICH [306][307][308]. Macrophages also monitor neuronal activity and impact neuronal structural remodelling in mice [309] and zebrafish [310], and thus may impact cerebral ECs indirectly via neurons.…”

A Multi-Model Pipeline for Translational Intracerebral Haemorrhage Research

“…We recognise that zebrafish larvae are developing animals, and different disease mechanisms may exist in comparison to adult humans. However, we have shown that key characteristics associated with the pathological response to blood in the brain are apparently conserved between young fish and adult humans, suggesting they can be reliably used to model aspects of ICH [87,88].…”

“…In addition, as recovery rates are so rapid in zebrafish larvae as demonstrated by Crilly et al, it poses questions on how this may differ to recovery in adult humans. However, understanding these types of processes following ICH during development may provide clues into how we might consider recovering the aged human brain in the future [87].…”

RNA-Seq Dataset From Isolated Leukocytes Following Spontaneous Intracerebral Hemorrhage in Zebrafish Larvae

“…Unlike more commonly used rodent models, ICH in bbh mutant zebrafish larvae can occur without the need for invasive surgical procedures and thereby mimic the spontaneous nature of the human condition more closely. We have previously characterized disease outcomes in the zebrafish bbh model which recapitulates key pathological outcomes observed in mammalian models and patients (Crilly et al, 2018(Crilly et al, , 2019. The secondary injury response in zebrafish larvae is identified by an increase in macrophage recruitment and activation within the head at 24 h post injury, whilst neutrophils are the most numerous leukocyte population at this developmental stage.…”