Zebrafish models of cerebrovascular disease

Abstract

Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens.

Figure 1

Phenotype comparison of zebrafish and human arteriovenous malformations (AVM). (A) In wild-type embryos (row 1), transient connections between the basal communicating artery (BCA) and primordial midbrain channel (PMBC) carry blood at 32 hpf but regress by 48 hpf (white arrows). In alk1 mutants (row two), one or both of these bilateral connections may be retained, forming an abnormal BCA–to–PMBC arteriovenous connection (white arrows). More posteriorly, lumenized connections drain the basilar artery (BA) to the primordial hindbrain channel (PHBC) in wild-type embryos at early times, but almost all regress by 48 hpf (row 3, white arrows). In alk1 mutants, one or more of these connections may be retained, forming a BA–to–PHBC AVM (row 4, arrows). This model resembles the human condition, seen in a digital subtraction cerebral angiogram. (B) In human AVMs, arterial branches (red arrows) connect directly to the venous circulation (blue arrows) through a high-flow fistula (purple arrow). One theory for AVM development is that they represent the abnormal persistence of normal transient developmental connections. Scale bars, 50 μm. Zebrafish images are two-dimensional confocal projections of Tg(kdrl:GFP)^la116; Tg(gata1:dsRed)^sd2 embryos, dorsal views, anterior leftwards. Endothelial cells are green; erythrocytes are magenta. Human digital subtraction angiogram is a lateral projection carotid artery injection in the late arterial phase. (Figure and legend modified from Corti P et al. Distributed under the terms of the Creative Commons Attribution (CC-BY) License).

Figure 2

Phenotype comparison of zebrafish and cerebral cavernous malformation. In an magnetic resonance imaging of a human (A), a cerebral cavernous malformation is apparent in the right frontal lobe (blue arrow). These lesions can result in catastrophic hemorrhage and/or seizure activity. Treatment with surgery is effective, and complete resection can be achieved (B) if lesions are in accessible areas. When lesions are deep or multiple, surgical treatment may not be indicated, underscoring the need to develop novel therapeutics. To understand the molecular mechanisms in the CCM pathway, zebrafish models of the disease have been generated using morpholino technology. Compared with control organisms (C and D), morphant knockdowns of rap1b (E and F), a gene that encodes a Ras GTPase effector protein for CCM1/Krit1, demonstrate disrupted endothelial junctions, resulting in intracranial hemorrhage (black arrows), similar to human lesions. Bars, 250 μM. (Figure modified from Gore A V et al. Distributed under the terms of the Creative Commons Attribution (CC-BY) License).